120 Theophylline: the Key for Bulls with Low In Vitro Blastocyst Rates?

Abstract

Although many efforts have been made to improve the sperm normal penetration ability for in vitro embryo production (IVP), sperm capacitation in bovine remains a bottleneck for in vitro fertilization (IVF). The variable responses of sperm to the different supplements in IVF medium makes the process suboptimal for every bull. Theophylline, a methylxanthine, has recently been reported to enhance and maintain in vitro blastocyst production due to prolonged motility and longevity of bovine sperm (Kang et al. 2015 J. Reprod. Dev. 61, 99-105). Here, we evaluated the effects of theophylline supplementation during IVF on developmental rates of bulls with known low and average blastocyst production under standard conditions. A total of 1498 cumulus–oocyte complexes were obtained by slicing from bovine ovaries. Cumulus-oocyte complexes were submitted to in vitro maturation (IVM) for 24 h. In vitro fertilization was performed for 19 h with (2.5 mM) or without theophylline using frozen–thawed sperm from 5 different bulls at a final sperm concentration of to 2 × 10⁶ cells mL⁻¹. Two of the bulls had a previous report of low blastocyst rates (A, B) and 3 showed an average blastocyst production (C, D, E). After IVF, presumptive zygotes were cultured in vitro for 8 days until the blastocyst stage. Cleavage and blastocyst rates were evaluated 3 and 8 days after IVF, respectively. R software (https://www.r-project.org/) was used to evaluate cleavage and blastocyst rates using a two-sample t-test. The supplementation of theophylline significantly increased blastocyst rates for bulls A, B, and E (Table 1; P < 0.05). The blastocyst rates did not differ for bull C and D with or without the use of theophylline (Table 1; P > 0.05). These preliminary results show that in vitro blastocyst production for low performance bulls can be improved by theophylline supplementation during IVF. The underlying mechanism probably involves phosphodiesterase (PDE) inhibition to increase intra-sperm/oocyte cAMP levels improving oocyte-sperm interaction.

Table 1.  Total oocytes and mean (± SEM) cleavage and blastocyst rates of bull sperm treated or not with theophylline

The authors thank Susanne Meese and Ulrich Witschi from Swissgenetics for the cooperation and donation of frozen bull sperm.