Effects of zinc chloride supplementation during …...Objectives •Determine the effects of adding...
Transcript of Effects of zinc chloride supplementation during …...Objectives •Determine the effects of adding...
Effects of zinc chloride supplementation during vitrification of ovarian tissue in pigs.
Emma Hicks
Cryopreservation
• Four Major Steps:• Addition of cryoprotectant• Freezing at low temperatures and storing in liquid N2
• Warming tissue• Thawing cells and removing cryoprotectants
(Adedelahi et al., 2013)
▪ Ovarian tissue cryopreservation preserves large numbers of oocytes versus other techniques.
(Adedelahi et al., 2013)
• Cryopreservation of ovarian tissue is conducted to help preserve the fertility of biomedical models.
(Mouttham & Comizzoli, 2016)
Follicular Damage and Stress• Ovarian tissue is susceptible to more damage during
vitrification due to various cell types and water permeability.
(Adedelahi et al., 2013)
▪ Vitrification increases reactive oxygen species in oocytes,leading to decreased viability.
(Gupta et al., 2010)
▪ Cryoprotectant agents can induce oxidative stress thus causing structural and functional changes in tissue.
(Tian et al., 2015)
• Zinc reduces oxidative stress by synthesizing proteins that are effective in reducing reactive oxygen species.
(Marreiro et al., 2017)
Objectives
• Determine the effects of adding 5 μg/mL 𝑍𝑛𝐶𝑙2 during vitrification on: in vitro follicle development
post-thawing fertilization success
embryonic development
Experimental Design
0 μg/mL
ZnCl2
0 μg/mL
ZnCl2
5 μg/mL
ZnCl2
5 μg/mL
ZnCl2
7 days
40-44
hours48
hours
6-8
hours
144
hours
12
hours
0 μg/mL
ZnCl2
5 μg/mL
ZnCl2
Semen Pellets
Fertilization
Characteristics
Cleavage Blastocyst
Ovary from
Cycling Gilts
Centrifuge Wash
Sperm
Concentration
Dilution
Oocyte WashThawing Media
Fertilization
Drops
Embryo
Culture
Ovary Cortex
Cross-Section 5 min5 min
5 min 5 min
Aspiration of
Follicles
Thawing Media
Follicle Evaluation
Primordial Primary Secondary Antral
• Single layer of
squamous cells
• Single layer of
cuboidal
cells/stratified
epithelium
• Theca interna
cells
• Granulosa cells
• Zona pellucida
• Antrum
• Cumulus cells
Damage Characteristics
Zona Pellucida
Disruption
No Defined
Antrum
Theca Interna
and Granulosa
Cell Disruption
Follicular Cells
in Cytoplasm
Follicle Counts
Treatment Primordial (%) Primary (%) Secondary (%) Antral (%)
0 μg/mL ZnCl2 52.7a 31.0a 18.6a 25.6a
5 μg/mL ZnCl2 39.4b 34.8b 14.2b 29.0b
Total Follicle Damage
Treatment Total Follicles (%) Damaged (%)
0 μg/mL ZnCl2 100 46.5a
5 μg/mL ZnCl2 100 23.2b
Type-Based Follicle Damage
0
10
20
30
40
50
60
70
80
90
100
Primordial Primary Secondary Antral
Perc
en
t O
ccu
rren
ce (
%)
Follicle Types
0 μg/mL Zinc Chloride
5 μg/mL Zinc Chloride
b b b ba a a a
a,b p < 0.05
Fertilization Characteristics
Media Supplementation % Penetrated
0 μg/mL ZnCl2 78.60 ± 12.60
5 μg/mL ZnCl2 54.50 ± 14.20
Media Supplementation % Polyspermic
0 μg/mL ZnCl2 71.40 ± 12.50a
5 μg/mL ZnCl2 27.30 ± 22.40b
Media Supplementation % MPN Formation
0 μg/mL ZnCl2 36.40 ± 13.70a
5 μg/mL ZnCl2 57.10 ± 11.10b
Sperm Penetration Rates (%)
Polyspermy Rates (%)
MPN Rates (%)
Embryonic Development
0
10
20
30
40
50
60
70
80
90
100
0 μg/mL Zinc Chloride 5 μg/mL Zinc Chloride
Cleavage
Blastocyst
a a a a
a,b p < 0.05
Conclusion• Supplementation of 5 mg/mL of ZnCl2
Improves follicle development:
Reduces incidence of follicular damage from vitrification
Improves follicular integrity
Increases antral follicle development
Improves post-thawing fertilization:
Reduces incidence of polyspermy
Increases male pronucleus formation
Questions?