Improvement of canine in vitro fertilization system using frozen-thawed sperm

Abstract

In vitro fertilization (IVF) in dogs has always been the main obstacle for researchers in opening up the possibility of preserving endangered species of dogs, understanding the inherited diseases between dogs and humans, and applying gene editing techniques. The first successful IVF with live puppies was reported towards the end of 2015, using fresh semen and in vivo matured oocytes combined with embryo freezing. A small number of studies have been performed in IVF using frozen-thawed sperm with in vivo/vitro matured oocytes, but low penetration and cleavage rates, delayed cleavage and high degenerated embryos were the main problems faced in this field. The post-thawing quality of sperm became one of several key elements in overcoming these problems. The aim of this study was to improve canine IVF using enhanced frozen-thawed sperm through a modified freezing protocol, antioxidant supplementation during cryopreservation, and adding conditioned media during capacitation. A multistep freezing protocol, comprising serial loading and dilution of cryoprotective agents by dividing the total volume of extender into 4 steps (14%, 19%, 27%, and 40%) every 30 sec, was compared to a single step method in sperm function, morphology and osmolytes content. A comparison of the effects of glycerol and ethylene glycol were also performed. A spermine treatment using 0, 0.1, 1, 5, or 10 mM was analyzed for its effect on sperm quality, reactive oxygen species (ROS) level, cryocapacitation rate and gene expression related oxidation. The conditioned media (CM) from human adipose-derived stem cells (ASCs) which was used as supplement in canine capacitating medium (CCM) during frozen-thawed sperm capacitation. The 0, 25 and 50% CM supplementation were compared and the viability and gene expression related fertility of those groups were determined. The optimum CM combination were used for IVF then cleavage rate and embryo transfer was evaluated. Frozen–thawed spermatozoa in the multistep group showed superior quality compared to those in the single step group, with regards to progressive motility, intactness of membrane and bend in tail. The multistep protocol also succeeded in minimizing osmolytes loss, such as carnitine and glutamate, compared to the single step group. Moreover, using glycerol with the multistep group was more advantageous in maintaining high sperm quality compared to using ethylene glycol. Although motility did not increase with spermine treatment, membrane integrity was significantly increased. Higher percentages of linearity and straightness with a lower amplitude of lateral head displacement (ALH) in the spermine treated group indicated that spermine inhibited hyperactivation. Concentrations of intracellular and extracellular ROS were decreased in the treatment groups. Higher expression of an anti-apoptotic gene (BCL2) and lower expression of a pro-apoptotic gene (BAX), together with decreased expression of mitochondrial ROS modulator 1 (ROMO1), DNA repair due to oxidative damage (OGG1), spermine synthase (SMS), NADPH oxidase associated with motility (NOX5) and spermine amino oxidase (SMOX), showed that 5 mM spermine treatment was beneficial for the spermatozoa. Furthermore, after thawing, the proportion of live spermatozoa with intact acrosomes in the treatment group was higher than in the control. After incubation of the spermatozoa in CCM, numbers of live capacitated spermatozoa with reacted acrosomes were higher in the spermine treated group than in the control. CCM supplemented with 25% CM resulted in a significantly higher percentage of motility, progressive motility, linearity and viability than control and 50% CM groups. The expression of gene related to DNA packaging, motility and fertility in the 25% CM group were significantly upregulated compared with the control group. The percentage of live sperm reacted acrosome in the treated group was also significantly greater than the control group. We collected the oocytes from 37 bitches and mature oocytes were recovered from 30 (81.1%) dogs then could produce 70.5% cleavage rate after IVF. Immature oocytes recovered from 3 bitches showed 25.0% cleavage rate and aging oocytes from 4 bitches (10.8%) showed 51.4% cleavage rate. Optimum cleavage rate were produced by IVF using mature oocytes compared with other stages. Moreover, IVF using frozen-thawed sperm resulted in a cleavage rate of more than 60%, which is higher compared to those of other studies. In conclusion, the multistep freezing method is superior at maintaining sperm function and osmolyte content. Spermine supplementation reduces ROS levels and decreases cryocapacitation, and adding 25% CM in CCM increases sperm motility, viability and fertility. Furthermore, an IVF system using enhanced frozen-thawed sperm can improve the canine embryo production.