Addition of D-penicillamine, hypotaurine, and epinephrine (PHE) mixture to IVF medium maintains motility and longevity of bovine sperm and enhances stable production of blastocysts <i>in vitro</i>

Abstract

The present study aimed to establish an efficient system for bovine embryo production by in vitro fertilization (IVF) that can achieve stable normal fertilization and blastocyst developmental rates in any bull without optimization of the sperm concentration in IVF medium. We examined the effects of a PHE mixture (20 μM D-penicillamine, 10 μM hypotaurine and 1 μM epinephrine), theophylline (2.5 mM), and sperm concentration (1, 2 or 5 × 106 cells/ml) on fertilization and blastocyst developmental rates. High cleavage rates (78.3 to 92.4%) and blastocyst developmental rates (31.9 to 62.0%) at day 7 were obtained in the presence of PHE and theophylline in IVF medium with a sperm concentration of 2 × 106 cells/ml using sperm from 9 bulls. In addition, the synergistic effect of PHE and theophylline on normal fertilization (2 pronuclei) was clarified at 12 h after IVF with a sperm concentration of 1 × 106 cells/ml. Moreover, high linearity, high flagellar beat cross frequency, and low amplitude of lateral head of motile sperm were found by computer-assisted sperm analysis. In conclusion, the combination of the PHE mixture and theophylline synergistically accelerates sperm motility and sperm penetration of bovine oocytes. Theophylline activates sperm motility with increasing intracellular cAMP. However, PHE prevents an excessive increase of cAMP and maintains sperm motility without hyperactivation. When the combination of PHE and theophylline is added to IVF medium at a sperm concentration of 2 × 106 cells/ml, we can achieve stable normal fertilization and blastocyst development in any bull.

Kinetic activity, membrane mitochondrial potential, lipid peroxidation, intracellular pH and calcium of frozen/thawed bovine spermatozoa treated with metabolic enhancers

“…In spite of the well‐known effects of the PHE in improving the oocyte penetration, the pronuclear formation and the efficiency of in vitro embryo production in cattle (Gonçalves et al ., ; Kang et al ., ), there are not evident effects on sperm quality after exposure to this mixture. PHE exposure does not affect the MMP as well as the integrity of sperm membranes and DNA (Gonçalves et al ., ).…”

Penicillamine prevents ram sperm agglutination in media that support capacitation

“…As agglutination occurs when trying to capacitate ram spermatozoa in TALP, it was important to assess if the inclusion of PEN in this medium would interfere in the capacitation process. It would seem unlikely as low doses of PEN (20 mM), in conjunction with hypotaurine and epinephrine (PHE), have been included in multiple bovine IVF protocols to improve oocyte penetration and embryo quality (Miller et al 1994, Goncalves et al 2014, Kang et al 2015. PEN has also been used in protein-free media to promote capacitation (Andrews & Bavister 1989, Keskintepe & Brackett 1996.…”

Antioxidant effects of penicillamine against in vitro‐induced oxidative stress in human spermatozoa

“…In frozen/thawed bovine spermatozoa, the mixture of penicillamine, hypotaurine and epinephrine (PHE mixture) improves mitochondrial membrane potential and promotes better maintenance of intracellular pH upon incubation at room temperature for 4 hr (Boni, Gallo, & Cecchini, 2017). Similarly, the combination of the PHE mixture and theophylline improves sperm motility and sperm penetration of bovine oocytes (Kang et al, 2015). Another study reported that although the IVF media supplementation with a heparin and PHE mixture affects bovine sperm quality, it contributes to sperm capacitation, due to an improvement of pronuclear formation and early embryonic development during bovine IVF procedures (Goncalves, Barretto, Arruda, Perri, & Mingoti, 2014).…”