14 Effects of long cooling periods of the ear skin on the isolation and cultivation of bovine fibroblasts for posterior use in cloning via nuclear transfer
Authors: Araujo J. M. , Oliveira R. A. , Cumpa H. C. B. , Cunha A. T. M. , Dode M. A. N. , Martins C. F. 
Published: December 2019

Many animals with high genetic value have died suddenly without having their germplasm conserved invitro. To simulate tissue transportation from the farm to the laboratory and to determine how long after an animal’s death it is possible to isolate and cultivate fibroblasts to form a cryobank for further use in nuclear transfer, ears of eight bovine females were obtained on the moment of death and preserved for 30 days at 5°C in a refrigerator. On Days 0, 2, 4, 7, 14, 21, and 30 postmortem (named D0 to D30), fibroblast cultivation was performed on Dulbecco’s modification of minimum essential media (DMEM) medium with 10% of fetal bovine serum. When in cellular confluence, cells were cryopreserved in DMEM with 10% of dimethyl sulfoxide. The following analyses were performed: time that allowed cell culture, time until initial cell attachment and proliferation, time until confluence was reached (counting from when the first cells attached and proliferated around the biopsies until they were confluent), contamination rates, cell concentration on freezing moment, and cell viability through membrane integrity via flow cytometry (using annexin and propidium iodide to verify viable, necrotic, and apoptotic cells). Test of means and Tukey’s test were used to compare data at 5%. All time points allowed cell isolation and culture. Contamination was more prevalent on Days 14, 21, and 30. Remaining results are presented in Table 1. While D0 cells took 4 days to initiate attachment and proliferation around the biopsies, and another 24.0±2.0 days to reach confluence, D30 cells needed 33.5±1.5 days to attach and proliferate and another 31 days to reach cellular confluence, totaling ~64.5±1.5 days from the day of cultivation until freezing against a total of 28±2.0 days for D0 cells. Concentration dropped drastically on Days 14, 21, and 30 and so did cell viability on Days 21 and 30. It was concluded that cooling of the bovine ear skin at 5°C is an important strategy for transporting bovine tissue for long distances and obtaining viable cells up to 30 days after an animal’s death. However, the increase in cooling time interferes with the proliferation and viability cell patterns until and after cryopreservation. The first cloned embryos from D30 cells were transferred to recipient cows to investigate any differences in embryo viability between different days. This second phase of the experiment is still ongoing.

Table 1.Mean and standard deviation of initial cell attachment/proliferation, time to confluence, concentration, and viability

Day
A/P (days)
Confluence (days)
Concentration (cell/mL)
Viability (%)

D0
4±0a
24±2a
1.334.375±131.375a
85.6±2.2a

D2
5.33±1.5a
28.0±3.1a
1.645.050±252.639a
82.19±16.81a

D4
4.33±1.03a
36±4b
1.571.656±234.462a
80.80±30.0a

D7
10.0±1.78b
36±0b
1.509.400±347.860a
75.02±28.52a

D14
19.6±2.19c
34.5±4.04b
698.125±131.203b
79.23±17.73a

D21
24.5±3.5c
36±0b
304.166±47.604c
27.46±12.46b

D30
33.5±1.5d
31±0b
311.250±0c
28.71±13.9b

a-dDifferent letters represent statistically significant differences (P&lt;0.05).