Yoshiyuki Ohta

For the conservation of endangered avian species, developing gamete preservation technologies is essential. However, studies in oocytes have not been widely conducted. In this study, assuming that the ovaries are transported to a research facility after death, we investigated the effect of ovary storage on oocytes for the purpose of cryopreserving avian female gametes by using a chicken as a model of endangered avian species. After excision, the ovaries were stored at either a low temperature (4 °C) or room temperature for 1–3 days. Ovarian follicles stored under different conditions for each period were examined by neutral red staining, histology, and gene and protein expression analysis. In addition, the pH of the storage medium after preserving the ovaries was measured. Then, ovarian tissues were vitrified to determine the cryopreservation competence. Storing the ovarian tissues at 4 °C kept the follicles viable and morphologically normal for 3 days with slow decline. In contrast, although different storage temperature did not influence follicle viability and morphology after only 1 day of storage, ovarian tissues stored at room temperature rapidly declined in structurally normal follicles, and viable follicles were rarely seen after 3 days of storage. Gene and protein expression analysis showed that apoptosis had already started on the first day, as shown by the higher expression of CASP9 under room temperature conditions. Furthermore, high expression of SOD1 and a rapid decline of pH in the storage medium under room temperature storage suggested the influence of oxidative stress associated with low pH in this condition on the follicle survivability in hen ovarian tissues. Our cryopreservation study also showed that ovarian tissues stored at 4 °C could recover after cryopreservation even after 3 days of storage. The described storage conditions and cryopreservation methods, which preserve chicken follicle survival, will lay the foundation of ovarian tissue preservation to preserve the fertility of wild female birds.

To explore metabolic characteristics during the post-hatch developmental period, metabolomic analyses of breast muscle and plasma were performed in chickens. The most significant growth-related changes in metabolite levels were observed between seven and 28 days of age. Some of these metabolites are essential nutrients or reported as growth-promoting metabolites. In the muscle, two imidazole dipeptides—carnosine and its methylated metabolite, anserine—increased with the development. These dipeptide levels may be, in part, regulated transcriptionally because in the muscle mRNA levels of carnosine synthase and carnosine methylation enzyme increased. In contrast, taurine levels in the muscle decreased. This would be substrate availability-dependent because some upstream metabolites decreased in the muscle or plasma. In branched-chain amino acid metabolism, valine, leucine, and isoleucine decreased in the muscle, while some of their downstream metabolites decreased in the plasma. The polyamines, putrescine and spermidine, decreased in the muscle. Furthermore, mRNA levels associated with insulin/insulin-like growth factor 1 signaling, which play important roles in muscle growth, increased in the muscle. These results indicate that some metabolic pathways would be important to clarify metabolic characteristics and/or growth of breast muscle during the post-hatch developmental period in chickens.

Low protein diets (LPs) constitute a reportedly effective form of nutritional therapy for canine chronic kidney disease and cirrhosis. These diets have long been feared to result in reduced muscle mass due to protein catabolism. This adverse effect, however, remains largely unrecognized in veterinary medicine as there are no easily applicable catabolism indicators. Therefore, we focused on urinary creatinine, a metabolite of protein in the urine, and examined whether its ratio to urinary urea nitrogen (UCrn/UN) can be used to assess protein catabolism. In Experiment 1, we first consecutively fed seven healthy beagles an LP, standard protein (SP), and high protein (HP) diet for 1 week each and then measured the UCrn/UN ratio at 2-h intervals from fasting to 16 h post-prandially. We consequently found that the UCrn/UN ratio was significantly elevated in the LP pre-prandially and at all post-prandial measurement points (P < 0.01). No significant differences were observed between the SP and HP. Analysis of fasting plasma amino-acid concentrations revealed that the concentration of methionine was significantly lower in the LP than in the other diets (P < 0.05). Although the effects of this change in amino-acid concentration were unclear, the UCrn/UN ratio was considered having increased due to a deficiency in protein and/or amino acids during LP feeding. In Experiment 2, we continuously fed five healthy beagles an LP for 18 weeks and then measured the UCrn/UN ratio as described above. We also measured changes in body composition with computed tomography. At weeks 10 and 18, the fasting UCrn/UN ratio was significantly higher than it was prior to the start of the LP; however, post-prandially, the UCrn/UN ratio decreased to the point that the significant difference disappeared. Muscle mass decreased at weeks 10 and 18. These results suggest that the fasting UCrn/UN ratio could be used as an indicator of protein catabolism in LP feeding. Our experiments thus indicate that examination of potential increases in the UCrn/UN ratio 1 week after introduction of LP feeding to healthy dogs could enable detection of body protein catabolism in long-term feeding of LP before muscle breakdown occurs.