BOEC-Exo Addition Promotes In Vitro Maturation of Bovine Oocyte and Enhances the Developmental Competence of Early Embryos.

Exosomes are nano-sized vesicles with abundant nucleic acids, proteins, lipids, and other regulatory molecules. The aim of this study was to examine the effect of BOEC-Exo on bovine in vitro oocyte maturation and in vitro embryo development. We found that a 3% Exo supplementation to IVM media significantly enhanced the oocyte maturation and reduced the accumulation of ROS in MII-stage bovine oocytes. Oocyte maturation related genes (GDF9 and CPEB1) also confirmed that 3% Exo treatment to oocytes significantly (p < 0.05) enhanced the oocyte maturation. Next, we cultured bovine cumulus cells and assessed the effects of 3% Exo, which showed a reduced level of apoptotic proteins (caspase-3 and p-NF-κB protein expressions). Furthermore, we examined the gap junction (CX43 and CX37) and cumulus cells expansion related genes (HAS2, PTX3, and GREM1) in cumulus-oocyte complexes (COCs), and all those genes showed significantly (p < 0.05) higher expressions in 3% Exo-treated COCs as compared with the control group. Moreover, peroxisome proliferator-activated receptors (PPARs) and lipid metabolism-related genes (CPT1 and FABP3) were also analyzed in both the control and 3% Exo groups and the results showed significant (p < 0.05) enhancement in the lipid metabolism. Finally, the oocytes matured in the presence of 3% Exo showed a significantly higher rate of embryo development and better implantation potential. Finally, we concluded that Exo positively influenced bovine oocyte in vitro maturation and improved the early embryo's developmental competence.

Melatonin Alleviates the Toxicity of High Nicotinamide Concentrations in Oocytes: Potential Interaction with Nicotinamide Methylation Signaling.

Despite the numerous studies on melatonin and nicotinamide (NAM, the active form of vitamin B3), the linkage between these two biomolecules in the context of signaling pathways regulating preimplantation embryo development has not yet been investigated. In this study, we used bovine oocyte model to elucidate the effect of melatonin on the developmental competence of oocytes under the stress of high NAM concentrations. Results showed that NAM (20 mM) administration during in vitro maturation (IVM) significantly reduced oocyte maturation and actin distribution, while induced reactive oxygen species (ROS) accumulation and mitochondrial dysfunction, the multiple deleterious effects that were alleviated by melatonin (10-7 M). The RT-qPCR and/or immunofluorescence showed upregulation of the apoptosis (Caspase-3, Caspase-9, and BAX), autophagy (Beclin-1, LC3A, LC3B, ATG7, LAMP1, and LAMP2), cell cycle (P21, P27, and P53), and DNA damage (COX2 and 8-OxoG) specific markers in oocytes matured under NAM treatment, compared to NAM-melatonin dual-treated and the untreated ones. In addition, the total cleavage and blastocyst development rate, as well as the total number of cells and the inner cell mass (ICM) per blastocyst, were reduced, while DNA fragmentation was induced, in the group of NAM sole treatment than NAM-melatonin cotreatment and control. Inspecting the underlying mechanisms behind NAM-associated toxicity revealed an increase in transcription pattern of NAM methylation (NNMT and AHCY) genes in NAM-treated oocytes while the opposite profile was observed upon melatonin supplementation. In conclusion, to our knowledge, this is the first study reporting that melatonin can protect oocytes and embryos from NAM-induced injury through its ROS-scavenging activity together with potential interaction with NAM methylation signaling.

Cardiopulmonary changes induced by retroperitoneal insufflation in healthy dogs in sternal recumbency with the abdomen unsupported.

This study assessed the effects of retroperitoneal carbon dioxide (CO2) insufflation on cardiopulmonary variables and intra-abdominal pressure (IAP) in mechanically ventilated dogs in sternal recumbency with the abdomen unsupported, following placement of a positioning kit and towels under the pectoral and pelvic regions. General anesthesia was induced in eight healthy adult male Beagles. A Swan-Ganz catheter was placed in the pulmonary artery via the jugular vein for cardiac output measurements. A Foley urethral catheter was placed to monitor transvesical IAP. A 10 mm balloon blunt-tip trocar was inserted into the retroperitoneal space. With a fixed respiratory rate and tidal volume by mechanical ventilation, insufflation pressure was sequentially increased from 0 to 10 mmHg in 5 mmHg increments, followed by desufflation. All variables were measured before insufflation, 5 min after the establishment of each insufflation pressure, and after desufflation. At 10 mmHg, the IAP was nearly equal to insufflation pressure. Cardiopulmonary function was not compromised at any point, although the cardiac index (CI), heart rate, mean arterial pressure (MAP), and mean pulmonary arterial pressure increased within normal ranges. End-tidal CO2 concentration, arterial CO2 partial pressure, and oxygen delivery index (DO2I) increased, whereas pH decreased, at 10 mmHg. CI, MAP, and DO2I did not recover to baseline after decompression. Thus, retroperitoneal CO2 insufflation up to 10 mmHg is well tolerated by mechanically ventilated dogs positioned in sternal recumbency with the abdomen unsupported, although sympathetic changes may occur with an insufflation pressure increase.

Feasibility of single-port retroperitoneoscopic adrenalectomy in dogs.

OBJECTIVE: To evaluate the feasibility of single-port retroperitoneoscopic adrenalectomy (SPRA) in dogs. STUDY DESIGN: A pilot experimental study. ANIMALS: Eight healthy beagle dogs. METHODS: SPRA was performed on the left and right sides (4 dogs each). Resection of the adrenal gland was performed through a SILS port using a retroperitoneal approach. Operative time was defined from skin incision to the completion of skin suture. Postoperative pain was evaluated by using 3 pain scores. Integrity of the adrenal gland capsule was evaluated by histologic assessment. RESULTS: Mean time taken to complete the SPRA was 44.1 minutes (range, 37-51) and was significantly longer on the right side than on the left side (P < .05). There were no complications intraoperatively or during 14 days of postoperative monitoring. The adrenal gland capsule was found to be injured in 3 of the 8 dogs by histologic assessment. CONCLUSION: This is the first report of SPRA in the veterinary literature. With this technique it is possible to perform adrenalectomy with some risk of capsule penetration and with excellent visibility. CLINICAL SIGNIFICANCE: This study suggests that SPRA is feasible and can be used to resect small adrenal tumors with minimal complications.

Bilateral medial iliac lymph node excision by a ventral laparoscopic approach: technique description.

The aim of this study was to describe a ventral laparoscopic technique for bilateral medial iliac lymphadenectomy in dogs. Twelve intact male purpose-bred research dogs, weighing less than 15 kg, were positioned in dorsal recumbency, and a 3-portal technique was used. Bilateral dissection was performed with vessel-sealing devices while tilting the surgical table by up to 30° towards the contralateral side of the target medial iliac lymph node (MILN) without changing the surgeon's position. Using a ventral laparoscopic approach, bilateral MILNs were identified and excised in all dogs. The mean times for unilateral and bilateral MILN dissections were 9.7 ± 3.8 and 21.0 ± 6.0 min, respectively. The mean times for the right and left MILN dissections were 10.8 ± 4.3 and 9.8 ± 2.5 min, respectively. The mean total surgery time was 43.7 ± 7.7 min. In total, 26 MILNs were dissected. Several complications, including mild to moderate capillary hemorrhage from perinodal fat and vessels (controlled laparoscopically), mild spleen trauma caused by the first trocar insertion and capsular damage of MILNs, were observed. However, there were no other major complications. All MILN samples were evaluated and deemed suitable for histopathologic diagnosis. Laparoscopic excision of MILNs is a useful method of excisional biopsy for histopathologic diagnosis. Using this ventral laparoscopic approach with the 3-portal technique, bilateral MILN dissection suitable for obtaining histopathologic samples could be achieved in a short time in dogs weighing less than 15 kg.

Retroperitoneal Laparoscopy in Dogs: Access Technique, Working Space, and Surgical Anatomy.

OBJECTIVE: To develop and describe a laparoscopic retroperitoneal access technique, investigate working space establishment, and describe the surgical anatomy in the retroperitoneal space as an initial step for clinical application of retroperitoneal laparoscopy in dogs. STUDY DESIGN: Cadaveric and experimental study. ANIMALS: Cadaveric (n=8) and healthy (n=6) adult dogs. METHODS: The retroperitoneal access technique was developed in 3 cadavers based on the human technique and transperitoneal observation. Its application and working space establishment with carbon dioxide (CO2 ) insufflation alone was evaluated in 5 cadavers by observing with a transperitoneal telescope and in 6 live dogs by repeated computed tomography (CT) scans at pressure of 0, 5, 10, and 15 mmHg. Recordings of retroperitoneoscopy as well as working space volume and linear dimensions measured on CT images were analyzed. RESULTS: Retroperitoneal access and working space establishment with CO2 insufflation alone were successfully performed in all 6 live dogs. The only complication observed was in 1 dog that developed subclinical pneumomediastinum. As pressure increased, working space was established from the ipsilateral to the contralateral side, and peritoneal tearing eventually developed. Working space volume increased significantly from 5 mmHg and linear dimensions increased significantly from 0 to 10 mmHg. With pneumo-retroperitoneum above 5 mmHg, retroperitoneal organs, including kidneys and adrenal glands, were easily visualized. CONCLUSION: The retroperitoneal access technique and working space establishment with CO2 insufflation starting with 5 mmHg and increasing to 10 mmHg provided adequate working space and visualization of retroperitoneal organs, which may allow direct access for retroperitoneal laparoscopy in dogs.