The effects of zinc on the maturation and fertilization of bovine oocytes

Brianna M. Price, Taylor F. Mittleider, Kayla Grau, and John Gibbons,
College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN

Zinc is an essential trace mineral in many species, playing many roles including an essential role in reproduction. Zinc is found throughout the body including the brain, kidney, liver, muscle, and bones where it plays a role in RNA and DNA metabolism (Hambridge and Krebs, 2007.) The highest concentrations of zinc are found in the eye and prostate gland (Hambridge and Krebs, 2007.) In the bovine oocyte, zinc is the most abundant transition metal with concentration fluctuations occurring during maturation and fertilization events (Que et al., 2019.) In all organisms examined, an event referred to as the “zinc spark” has been documented as an essential reproductive phenomena (Que et al., 2019.) High concentrations of zinc are present in the female gamete prior to the zinc spark, where zinc is released from the oocyte following intracytoplasmic sperm injection and natural encounters with a sperm cell (Bernhardt et al., 2012; Duncan et al., 2016; Que et al., 2019.) Higher quantities of zinc release during this event has been associated with higher quality embryos (Duncan et al., 2016; Picco et al., 2010; Zhang et al., 2016.) Zinc has been shown to play an important role in DNA stabilization during the fertilization process, when DNA is in a haploid state, including protection from damage and apoptosis (Anchordoquy et al., 2014.) The ability to produce in vitro bovine embryos provides an ideal model to enhance understanding of fertilization events in human reproduction. This studied examined the role of zinc in in vitro maturation and fertilization of bovine oocytes and tested the hypotheses that dose dependent zinc supplementation would enhance oocyte maturation and the chelation of zinc would inhibit fertilization and early embryonic development.

Evaluation of Zinc Supplementation on In Vitro Maturation
Bovine oocytes were obtained via follicular aspiration of postmortem ovaries harvested from an abattoir. Selected oocytes contained at least three layers of cumulus cells and a homogenous cytoplasm. Oocytes were separated into four in vitro maturation treatment groups supplemented with 0, 5, 10, and, 20 μM zinc. Supplementation doses where determined from analysis of zinc concentrations in adult cow plasma and follicular fluid (10.55 μM and 11.47 μM, respectively) and commercial maturation and fertilization medias (1.07 μM for each). Oocytes were considered mature if they had reached Metaphase II and had expelled their first polar body after 18 hours in maturation media. There was no statistical significance found in the maturation rates of the oocytes (78.1 ± 3.0%, 59.5 ± 4.3%, 69.8 ± 7.7%, 62.3 ± 3.2%, respectively). Mature oocytes were statistically analyzed by Chi Square test.

Evaluation of Zinc Chelation on In Vitro Fertilization and Embryo Development
The effects of zinc chelation on fertilization was observed in oocytes matured in 0 μM zinc, fertilized with frozen-thawed bull semen of a characterized bull, and separated into two groups. A zinc chelated group contained 2.7 mM of TPEN (tetrakis(2-pyridinylmethyl)-1-2-ethanediamine) supplemented in the fertilization media compared to non-treated controls. Following fertilization, the presumptive zygotes were cultured in their respective groups for 7 days (no TPEN). Embryonic development to the morula or blastocyst was analyzed by Chi Square test. The TPEN treated group had a statistically lower cleavage rate (p<0.05) than the control group (46.1 ± 2.3% and 75.6 ± 3.4%, respectively). Embryo development rate to morula stage was also statistically lower (p<0.05) in the TPEN treated group compared to the controls (15.4 ± 0.03% and 37.8 ± 0.03%, respectively). The average embryo developmental stage scores analyzed by ANOVA were significantly lower (P<0.001) in the TPEN treated group compared to the controls (2.2 ± 0.1 and 3.4 ± 0.2, respectively).

This study supports the concept that zinc supplementation has minimal effects on in vitro maturation of oocytes; however, removing zinc during in vitro fertilization, significantly decreased cleavage rate and embryo development to blastocyst. Future studies may determine a more precise role of Zinc during sperm penetration and fertilization mechanisms.


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