Mammary gland tumors are the most common canine neoplasms. They account for 25–50% of all tumors diagnosed in bitches. Metastases and recurrences develop in about 35–70% of bitches following excision. The presence of regional lymph node metastases is a relevant factor affecting prognosis and treatment in cases of mammary gland tumors. The sentinel lymph node (SLN) is the first lymph node (or nodes) in the regional lymphatic basin that receives lymphatic flow from the primary neoplasm. The aim of this study is to investigate the SLN with indirect lymphography for a mammary tumor in dogs. The knowledge of the precise drainage pattern and SLN of the neoplastic mammary glands would provide clinically relevant information to the surgeon and to the oncologist, and it would be of high importance for the surgeon not only for performing the most adequate surgical excision but also for determining an accurate post-surgical prognosis.

Juvenile in vitro embryo technology (JIVET) provides exciting opportunities in animal reproduction by reducing the generation intervals. Prepubertal oocytes are also relevant models for studies on oncofertility. However, current JIVET efficiency is still unpredictable, and further improvements are needed in order for it to be used on a large-scale level. This study applied bioengineering approaches to recreate: (1) the three-dimensional (3D) structure of the cumulus–oocyte complex (COC), by constructing—via bioprinting technologies—alginate-based microbeads (COC-microbeads) for 3D in vitro maturation (3D-IVM); (2) dynamic IVM conditions, by culturing the COC in a millifluidic bioreactor; and (3) an artificial follicular wall with basal membrane, by adding granulosa cells (GCs) and type I collagen (CI) during bioprinting. The results show that oocyte nuclear and cytoplasmic maturation, as well as blastocyst quality, were improved after 3D-IVM compared to 2D controls. The dynamic 3D-IVM did not enhance oocyte maturation, but it improved oocyte bioenergetics compared with static 3D-IVM. The computational model showed higher oxygen levels in the bioreactor with respect to the static well. Microbead enrichment with GCs and CI improved oocyte maturation and bioenergetics. In conclusion, this study demonstrated that bioengineering approaches that mimic the physiological follicle structure could be valuable tools to improve IVM and JIVET.

The genotoxic and nephrotoxic mycotoxin Ochratoxin A (OTA) has also been reported to have adverse effects on oocyte maturation and embryo development. Previous studies on the effects of OTA on female fertility have used micromolar concentrations, but no information is available to date on effects in a more relevant nanomolar range. This study used a juvenile sheep model to evaluate the effects of oocyte exposure to low levels of OTA on maturation, fertilization, and embryo development. Further, it was investigated whether different mechanisms of action of OTA could be responsible for varying toxic effects at different levels of exposure. Cumulus-oocyte-complexes (COCs) were exposed to 10 μmol/L–0.1 nmol/L OTA during in vitro maturation and evaluated for cumulus viability, oocyte maturation, and bioenergetic/oxidative status. COCs were subjected to in vitro fertilization, embryo culture, and embryo quality assessment via morphology, viability, bioenergetic/oxidative status, and time-lapse monitoring. At micromolar concentrations, OTA induced cytotoxic effects, by reducing cumulus expansion and oocyte maturation. OTA altered temporospatial dynamics of zygote pronuclear formation and embryo morphokinetics. Blastocysts, even morphologically normal, were found to undergo collapse events, which were probably related to boosted blastocyst mitochondrial activity. At nanomolar concentrations, OTA did not affect COC morpho-functional parameters, but impaired oocyte ability to prevent polyspermy and increased blastocyst apoptosis. In conclusion, in the female germ cell, cytotoxic nonspecific effects characterize OTA-induced toxicity at high exposure levels, whereas fine tuning-mode effects, not associated with altered cell viability and integrity, characterize OTA toxic action at low levels.

This study aimed to evaluate donkey seminal plasma metabolites and relate this information to the main characteristics of sperm quality. Sperm kinetics from 10 donkey stallions were analyzed with a computerized system at the time of collection (T0) and after 24 h storage at 4 °C (T24). Seminal plasma was frozen at −80 °C for subsequent proton nuclear magnetic resonance (¹H NMR) spectroscopy. On three stallions, semen collection was repeated monthly for three times and sperm analysis also included mitochondrial activity and oxidative status. One stallion was azoospermic and a second semen collection was performed after one month. In the seminal plasma, 17 metabolites were identified; their levels showed numerous significant variations between the azoospermic and the normospermic individuals and grouped in well-defined clusters in a multivariate analysis. Comparing individuals with high and low sperm motility, the only discriminating metabolite was phenylalanine, whose levels were lower in the latter, as in the azoospermic individual. Phenylalanine was also the only metabolite highly correlated with all sperm kinematic parameters at T24. In conclusion, the present study has provided relevant information on the chemical characteristics of donkey semen, identified relationships between seminal metabolites, semen parameters, and sperm kinetics, and offered insights for future technological applications.

In conventional assisted reproductive technologies (ARTs), oocytes are in vitro cultured in static conditions. Instead, dynamic systems could better mimic the physiological in vivo environment. In this study, a millifluidic in vitro oocyte maturation (mIVM) system, in a transparent bioreactor integrated with 3D printed supports, was investigated and modeled thanks to computational fluid dynamic (CFD) and oxygen convection-reaction-diffusion (CRD) models. Cumulus-oocyte complexes (COCs) from slaughtered lambs were cultured for 24 h under static (controls) or dynamic IVM in absence (native) or presence of 3D-printed devices with different shapes and assembly modes, with/without alginate filling. Nuclear chromatin configuration, mitochondria distribution patterns, and activity of in vitro matured oocytes were assessed. The native dynamic mIVM significantly reduced the maturation rate compared to the static group (p < 0.001) and metaphase II (MII) oocytes showed impaired mitochondria distribution (p < 0.05) and activity (p < 0.001). When COCs were included in a combination of concave+ring support, particularly with alginate filling, oocyte maturation and mitochondria pattern were preserved, and bioenergetic/oxidative status was improved (p < 0.05) compared to controls. Results were supported by computational models demonstrating that, in mIVM in biocompatible inserts, COCs were protected from shear stresses while ensuring physiological oxygen diffusion replicating the one occurring in vivo from capillaries.