Improved embryo development using high cysteamine concentration during IVM and sperm co-culture with COCs previous to ICSI in bovine

Abstract

In contrast to other species, intracytoplasmic sperm injection (ICSI) in bovine remains inefficient, resulting in low embryo developmental rates. It is unclear whether such inefficiency is due to the poor response of bovine ooplasms to the injection stimulus, or to the inability of bull sperm to induce oocyte activation. In order to facilitate these events, two strategies were assessed: the use of high concentration of cysteamine [Cys] during IVM; and the selection of sperm attached to cumulus cells after incubation with COCs for ICSI. First, COCs were IVM with increasing [Cys] and subjected to IVF. Zygotes from all groups were cultured under different O tensions and development to blastocyst was evaluated. In a second experiment, sperm were co-cultured for 3 h with COCs and acrosome reaction was studied. Afterwards, the best IVM and IVC conditions determined on Experiment 1 were used for ICSI assay. COCs were matured for 21 h with 1 (Cys 1) or 0.1 mM Cys (Cys 0.1 groups, standard condition). In addition, COCs were incubated for ≥3 h with 16 × 10 sperm/ml and only sperm attached to cumulus cells were selected for ICSI (ICSI + Co-cult groups). After chemical activation, embryos were cultured in SOF medium under low O tension. Cleavage and blastocyst rates were evaluated at days 2 and 7 of IVC, respectively. Finally, the relative expression of eight genes indicators of embryo quality was compared between ICSI and IVF control blastocysts by qPCR. Cleavage rates were higher for Cys 0.1 ICSI + Co-cult and Cys 1 ICSI + Co-cult groups (n = 117, 92% and n = 116, 79%, respectively) compared to their controls (n = 132, 60% for Cys 0.1 ICSI and n = 108, 52% for Cys 1 ICSI) (p ≤ 0.05). Interestingly, the combined treatment (Cys 1 ICSI + Co-cult) showed higher blastocyst rates than all other ICSI groups (23 vs. 11, 18 and 14% for Cys 0.1 ICSI + Co-cult, Cys 1 ICSI, and Cys 0.1 ICSI, respectively) (p ≤ 0.05). Moreover, incubation with COCs increased the rates of live acrosome reacted sperm (p ≤ 0.05). The relative abundance of mRNAs coding for INFτ, CAT, DNMT1, OCT4, and HDAC3 did not differ between treatments (p ≤ 0.05). SOD2, HADC1 and HADC2 expression was higher for Cys 0.1 ICSI than for IVF embryos (p ≤ 0.05). Group Cys 1 ICSI did not differ from IVF for those three genes, neither did Cys 1 ICSI + Co-cult, except for HDAC1 (p ≤ 0.05). In conclusion, the use of 1 mM Cys during IVM and of sperm incubated with mature COCs might be a good strategy to improve ICSI outcomes in cattle.

Cysteamine in Maturation Medium Enhances Nuclear Maturation and Fertilization Rate of Sheep Oocytes In Vitro

“…These data indicated that cysteamine supplementation was presumable could increase GSH concentration either for protection from ROS or to improve fertilization rate. This is confirmed by Canel et al (2018) that cysteamine supplementation during IVM, competent oocytes could increase their ability to synthesize GSH, facilitating the replacement of sperm protamins by histones during the male pronuclear formations. Although cysteamine in concentration 50 µm could not increase nuclear maturation, however it is presumable could increase oocyte quality, thus improve fertilization rate.…”

Exogenous activation and inhibition of plasminogen/plasmin activity during in vitro maturation of bovine cumulus‐oocyte complexes: A biological and spectroscopic approach

“…After IVM, about 10 matured COCs per drop were co‐incubated with sperm for 5 h at 38.5°C under 5% CO 2 in humidified air. This IVF protocol using a high sperm concentration and a short period of gametes co‐incubation was the same used by other authors (Canel et al, 2018; Hiriart et al, 2013; Vichera et al, 2011) and the one we used in previous studies (Barrera et al, 2018; García et al, 2015) obtaining results within standard parameters. Following the IVF period, presumptive zygotes were harvested from the insemination droplets; their CCs were removed by repeated pipetting and then washed three times with H‐TALP.…”

“…After IVM, about 10 matured COCs per drop were co-incubated with sperm for 5 h at 38.5°C under 5% CO 2 in humidified air. This IVF protocol using a high sperm concentration and a short period of gametes co-incubation was the same used by other authors (Canel et al, 2018;Hiriart et al, 2013;Vichera et al, 2011) and the one we used in previous studies (Barrera et al, 2018;García et al, 2015) | 69 n = 150; IVM-C: n = 150) as previously described (García et al, 2016).…”

Bull spermatozoa selected by thermotaxis exhibit high DNA integrity, specific head morphometry, and improve ICSI outcome

“…These results suggest that thermotaxis might have been able to select spermatozoa with the ability to activate the oocyte or decondense the sperm head properly after cytoplasmic injection. We hypothesized that spermatozoa selected by thermotaxis could be properly capacitated and this could be the reason why oocyte activation was not necessary to achieve similar or even higher embryo production rates in comparison to other studies that used ICSI with oocyte activation [50][51][52]. These results are prominent for future studies to optimize the current methodology for bull sperm selection by thermotaxis, which in combination with ICSI and oocyte activation might increase the success of ICSI in this species.…”