127 l-Carnitine Supplementation During In Vitro Maturation Improves Developmental Competence of Canine Oocytes

Abstract

In vitro embryo production (IVP) in the domestic bitch is important for conservation of endangered canids. Compared with various domestic animals, the development of assisted reproductive technologies (ART) in the dog has lagged behind, mainly due to the low percentage of oocytes that can reach metaphase II (MII) stage after in vitro maturation (IVM). Beneficial effects of l-carnitine (LC) on embryonic development in culture have been reported in many mammalian species; however, no studies have been conducted in dogs. The aim of the present study was to investigate the effect of LC supplementation during IVM of canine oocytes on nuclear maturation, fertilization status, and pre-implantation development following IVM/IVF. Cumulus-oocyte complexes (COC) were collected by slicing ovaries obtained from dogs (n = 20, 1 to 6 years of age) after ovariohysterectomy. The COC were subjected to IVM for 72 h in a medium (TCM-199) supplemented with LC at different concentrations (0.1, 0.3, 0.6, 1.0, or 2.0 mg mL⁻¹) or without LC supplements (0 mg mL⁻¹; control). Matured oocytes were fertilized in vitro with frozen–thawed spermatozoa, and presumptive zygotes were cultured in SOF medium for 7 days. Frequencies of nuclear maturation (72 h post-IVM), fertilization rates (18 h post-insemination), and embryo development (Days 2 to 5 post-insemination) were evaluated. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparisons test. Supplementation of IVM medium with 0.3 or 0.6 mg mL⁻¹ LC significantly improved (P ≤ 0.05) maturation (35.4% and 41.4%) and fertilization (21.3% and 25.8%) rates compared with the controls and with other LC-supplemented groups; values ranged from 20.1% to 25.0% for maturation and from 12.1% to 14.6% for fertilization. Cleavage (2- to 16-cell stages) was significantly higher (P ≤ 0.05) in the 0.6 mg mL⁻¹ LC supplemented group than the 0.3 mg mL⁻¹ supplemented group (16.3% v. 13.3%). These values were significantly higher (P ≤ 0.05) than those in other groups. Interestingly, 4.5% of IVM/IVF oocytes were developed to morula in 0.6 mg mL⁻¹ LC supplemented group which was significantly higher (P ≤ 0.05) than those developed in the 0.3 mg mL⁻¹ supplemented group (1.0%). No embryos developed beyond the 2- to 16-cell stage in the rest of the groups. In conclusion, l-carnitine supplementation during IVM is particularly efficient in improving nuclear maturation and pre-implantation embryo development of canine oocytes after IVF. These outcomes are important for the improvement of IVM conditions that can advance the efficiency of ART in dogs.