RESEARCH ARTICLE
J Res Vet Med. 2021: 40 (2) 131-135
DOI: 10.30782/jrvm.1027065
Biomechanical Comparison of The Effects of The Storage Temperature 
on Tibiotarsus in Japanese Quail
 Bayram Suzer*
Department of Anatomy, Faculty of Veterinary Medicine, Bursa Uludag University, Nilufer/Bursa, 16059, Turkey
Received 22-11-2021 Accepted 18-12-2021
Abstract
The study aimed to compare the effects of different cryopreservation temperatures on mechanical properties and determine the optimal 
cryopreservation temperature for bones in Japanese quail. Bone biomechanical tests are getting more attention but, fresh bones are not always 
available for testing and have a limited lifespan. Cryopreservation of biological specimens is often needed during tissue preparation and me-
chanical testing. In the study, the tibiotarsi were collected from 8 weeks of age quail, and bones were divided into four groups of fresh bones; 
frozen at 0 oC, frozen at -20 oC, and frozen at -80 oC. Frozen bones were kept in the freezer for three weeks. After three weeks, bones were 
subjected to a three-point bending test for biomechanical evaluation. There was no significant difference between the mechanical strength 
properties of fresh tibiotarsi and the tibiotarsi stored in three different storage conditions of 0 oC, -20 oC, or -80 oC. It was observed that cryo-
preservation of tibiotarsi at 0, -20, and -80 °C for up to three weeks did not negatively affect bone biomechanical properties in quail.
Keywords: Biomechanics, cryopreservation, freezing, storage temperature.
Introduction sues, as it is impossible to perform tests in vivo or immedi-
ately after sacrifice10. Therefore, cryopreservation of tissue 
Over the past decades, bone-breaking strength received samples before mechanical testing has become an accepted 
more attention. Bone measurements such as bone fracture technique11. However, it is still criticized whether preserva-
strength, bone ash, bone ash concentration, bone mineral tion methods affect bone properties or bone strength.
content, and bone density have been used as crucial indi- Researchers have conducted several studies to understand 
cators of bone status in poultry1-6. It is often impractical the effects of freezing and thawing on bone mechanical 
to test freshly harvested bone7. On the other hand, fresh properties on various species such as chickens9, dogs11, 
bones are not always available, have a limited lifespan (ap- mice12, rats13, pigs14, and deer15. In recent years, the use of 
prox. three weeks), and have difficulties in shipping or han- Japanese quail (Coturnix coturnix japonica) in biomedical 
dling8. Cryopreservation of biological specimens is often research has increased16, and its use in biological, genetic, 
unavoidable during tissue preparation and mechanical aging, and disease research has become widespread7. How-
testing. In particular, it becomes difficult to immediately ever, it is not known about the effects of freezing at differ-
process bone samples to measure bone strength and oth- ent temperatures on bone properties in quail. Therefore, 
er parameters with large numbers of animals. Therefore, if this experiment aimed to compare the effects of different 
large amounts of bone cannot be processed simultaneously, storage temperatures on some mechanical properties and 
an effective procedure is required to preserve and stabilize determine the optimal cryopreservation temperature for 
bone mineralization in the bone9. Postmortem storage has future bone biomechanical tests in Japanese quail. 
been used for biomechanical measurement of living tis-
 * Corresponding author:   Bayram Suzer, Department of Anatomy, Faculty of Veterinary Medicine, Bursa Uludag University, Nilufer/
Bursa, 16059, Turkey, +902242940867, suzer@uludag.edu.tr
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Suzer 2021
Material and Methods the load was applied to the midpoint of the bone length 
Animals and Bone Collection at the middle of the span. The loading was applied with 
The tibiotarsi were collected from eight weeks of age quail a constant speed of the load head until the bones broke. 
raised for commercial production (slaughtering material) Ultimate load (Fmax)was read as the highest load from 
in Animal Health and Animal Production Research and the load-displacement curve. Subsequently, mid-shaft sec-
Application Center in Bursa Uludag University Faculty tions of tibiotarsus were obtained from the fracture site 
of Veterinary Medicine. The quail were divided into four with a wire saw, and sections were photographed under a 
groups, and a total of twenty-four quail were used, with stereomicroscope (Motic, Model: SMZ-168, Hong Kong). 
six quail in each group. Experimental groups were: fresh Solidworks R17 3D CAD software (Dassault Systèmes, 
bones; frozen at 0 oC (Nuve ES120, NUVE, Ankara, Tur- Waltham, MA; USA) was used for determining the cortical 
key); frozen at -20 oC (Bosch KSU3921NE/01, Robert Bosch area (Acort) and the minimum principal moment of iner-
GmbH, Gerlingen, Germany), and frozen at -80 oC (Forma tia (Imin). Young’s modulus or modulus of elasticity (E) 
88000 Series, Thermo Scientific, Massachusetts, United was calculated by using the following equation:
States). After sacrifice, the bones were cleaned surround- E=(FL3)/(48δImin)
ing soft tissues. Before freezing, the bones were wrapped Imin is the minimum principal moment of inertia, F is the 
in physiological saline (0.9% NaCl) solution-soaked gauze ultimate load, L is the length between support, δ is the dis-
and bagged to prevent drying and tagged17. Then, the bones placement under the corresponding force.
were frozen at different temperatures (0, -20, and -80 oC) 
until mechanical tests and cortical area analysis were con- Statistical analysis
ducted. As the correlation coefficient for the breaking strength of 
right and left tibias of broiler chickens is at least 0.90 for 
Freezing and thawing procedure fresh bones20, the paired bone technique was used, and left and right tibiotarsi were combined to conduct the statisti-
1. The fresh group consisted of fresh bones, and mechan- cal analysis. Statistical analyses were performed with IBM 
ical tests were performed at room temperature within SPSS (SPSS, Version 23.0; Chicago, IL). Data were tested 
45 minutes after bone collection. for normality of distribution and homogeneity of varianc-
2. The bones in the 0 oC group were stored in sealed plas- es, and one-way analysis of variance (one-way ANOVA) 
tic bags at 0 oC for 3 weeks, then thawed at 37 oC in was used for statistical evaluation.
an oven (Nuve ES120, NUVE, Ankara, Turkey) for 1h 
and to be followed by the mechanical test at room tem- Results
perature. Results of the mechanical three-point bending test and 
3. The bones in the 20 oC group were stored in sealed bone properties are presented in Table 1. As a result of 
plastic bags at -20 oC for 3 weeks, then they were de- three-week freezing, there was no significant difference 
frosted at 37 oC in an oven (Nuve ES120, NUVE, An- between the mechanical strength characteristics (ultimate 
kara, Turkey) for 1h, and the mechanical test was per- load, minimum moment of inertia, Young’s modulus, cor-
formed at room temperature after thawing. tical area) of fresh tibiotarsi and the tibiotarsi stored in 
4. The bones in the 80 oC group were stored in sealed three different storage conditions of 0 oC, 20 oC or 80 oC. 
plastic bags at -80 oC for 3 weeks, then they were de- Likewise, no significant differences were observed between 
frosted at 37 oC in an oven (Nuve ES120, NUVE, An- different storage conditions (PFmax=0.657; PImin=0.393; 
kara, Turkey) for 1h, and the mechanical test was per- PE=0.731; PAcort= 0.317). 
formed at room temperature afterward13. Table 1. Mechanical properties of tibiotarsus measured after different 
storage methods
Mechanical Testing 
A three-point bending test was performed on each tibi-
otarsus of quail after the freeze-thaw procedure.  A cus-
tom-made testing machine, designed by Dr. Kenan Tufek-
ci, according to Tufekci et al.18, was used to measure force 
and corresponding displacement for low-strength materi-
als. The tests were performed at a constant loading head 
speed of 10 mm/min19. The average tibiotarsus length was *Data were presented as Mean±SEM (Standart Error of Mean)
49.44 ± 1.56 mm; therefore, the span between supports *Significance was assessed at the level of P< 0.05.
was adjusted to 40% of the total bone length, 20 mm, and Fmax: Ultimate load, Imin: Minimum moment of inertia, E: Young’s modu-
lus, Acort: Cortical area.
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Suzer 2021
Discussion searchers brought a perspective to this effect and suggested 
Especially in large-scale biomechanical studies, it is not al- a possible reason for differences in mechanical properties 
ways possible to work with fresh bones immediately after of the bone is the formation and enlargement of ice-crys-
animal sacrifice. Therefore, researchers used several storage tals37. When the bones are frozen at -20 °C, the bones slow-
methods at different temperatures at various times. Unlike ly lose moisture due to evaporation38, which increases the 
most studies, an avian species, quail, was used instead of a size of the ice crystals and causes structural damage to the 
mammalian species in the present study. Park et al.⁹ and bone tissue39. Lee and Jasiuk37 also suggested that cellular 
Lott et al.21 also investigated the effects of cryopreserva- enzymes could be another reason for bone degradation. 
tion on the mechanical properties of chicken bones. In the The enzymes that degrade the organic matrix are still effec-
present study, quail was selected because its importance tive at -20 °C. To avoid cellular destruction, temperatures 
has increased in biomedical studies in recent years16. The of -70 degrees and below should be preferred for long-term 
experiment was designed to examine the effects of freezing storage conditions37.
at 0°C, -20°C, and -80 oC on some biomechanical proper- In conclusion, it was observed that cryopreservation of ti-
ties of the tibiotarsus in quail. This experiment is also the biotarsi at 0, -20, and -80 °C for up to three weeks did not 
first study comparing the bone biomechanical properties negatively affect bone biomechanical properties in quail. 
of quail tibiotarsus at different cryopreservation tempera- Therefore, researchers can use 0, -20, and -80 °C tempera-
tures. tures as a convenient preservation method in quail species. 
Researchers used different temperatures between -18 °C In future studies, by planning a study longer than one year, 
and -70 °C for various periods before testing specific bio- comparing the bone mechanical properties is recommend-
mechanical properties of human and animal bones, such ed to examine the long-term effects of cryopreservation 
as rigidity and elasticity22 bending, torsion, toughness, and temperature.
stiffness23 and ultimate load24,25. Based on the previous lit-
erature, changes in bone biomechanical properties were Conflict of interest
assessed with various freezing or cooling storage methods The author declares no conflict of interest.
by using compression and bending tests26. Literature sug-
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