Repeated Freeze-Thaw Cycles but Not Short-Term Storage of Fecal Extracts at Ambient Temperature Influence the Stability of Steroid Metabolite Levels in Crested Macaques

Abstract

The objective of this study was to examine the effects of repeated freeze-thaw cycles and short-term storage of fecal extracts at ambient temperature on the stability of fecal glucocorticoid (fGCM) and estrogen metabolite (fEM) levels from crested macaques.In total 100 aliquots of fecal extracts from fecal samples collected from female crested macaques (Macaca nigra) living at the Tangkoko-Batuangus Nature Reserve, North Sulawesi were used. We performed two different experiments: (1) An experiment to investigate if levels of fGCM and fEM measured from fecal extracts that were exposed to two, four, six and eight repeated freeze-thaw cycles (test groups) differ to control samples (i.e. fecal extracts always stored frozen); (2) An experiment to evaluate whether storing fecal extracts at ambient temperature for two, four, six, and eight days (test groups)affects the levels of fGCM and fEM compared to the control group (i.e. fecal extracts frozen immediately).Results showed that hormone levels were significantly increased (P<0.05) after four freeze-thaw cycles for fGCM and after eight freeze-thaw cycles for fEM. By contrast, there was no significant difference (P>0.05) in levels of fGCM and fEM between the test groups and the control group in fecal extracts stored at ambient temperature. In conclusion, our data show that more than two and six repeated freeze-thaw cycles should be avoided when measuring fGCM and fEM in crested macaque fecal extracts, respectively. We also demonstrate that storing fecal extracts at ambient temperature is possible for at least 8 days without taking a risk of affecting the stability of fGCM and fEM levels.

The validation of a commercial enzyme-linked immunosorbent assay and the effect of freeze-thaw cycles of serum on the stability of cortisol and testosterone concentrations in Aceh cattle

“…A paired t-test was used for C, whereas for T, an independent t-test was used. For the freeze-thaw cycles experiments, the proportion of change in C and T concentrations relative to the control was calculated as (a n -x n /x n ) x 100, where a n is the nth sample value in each freeze-thaw cycles series (two, four, six, eight times) and x n is the value at time zero (control) of the nth sample 8, 16 . The C data were normally distributed (p>0.05), whereas the T data was not (p<0.05).…”

“…These findings show that T is more resilient to freeze-thaw cycles compared to C. The reason why C concentrations decreased after exposure to repeated freeze-thaw cycles and T concentrations did not, it is not entirely clear. For C, it is possible, however, that the serum left at high temperatures (27.6±0.7°C) during the six hours of the thawing process might facilitate the increased degradation of C 16 . Consequently, less antigen in the serum binds with the antibody of the assay 38 .…”

“…frequent power outages) should be avoided. Some efforts can be performed to prevent the possibility of repeated freeze-thaw cycles 16 . First, installing a backup generator to supply electricity when there are power outages.…”

“…Another critical issue for hormone measurements is the repeated freezing and re-thawing of the samples (freeze-thaw cycles) during the storage prior to the analyses 16 . Freezing the serum or plasma at -20°C or lower is an ideal storage method 17 .…”

The validation of a commercial enzyme-linked immunosorbent assay and the effect of freeze-thaw cycles of serum on the stability of cortisol and testosterone concentrations in Aceh cattle

“…Another critical issue for hormone measurements is the repeated freezing and re-thawing of the samples (freeze-thaw cycles) during the storage prior to the analyses 15 . Freezing the serum or plasma at -20°C or lower is an ideal storage method 16 .…”