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Volume 40, No. 3 May/June 2019
ISSN 0143-2044
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Hyaluronidase
pretreatment improves the cryopreservation of human ovarian tissue
Zhun Xiao, Zhong-Ying Huang, Yao-Yao Zhang, Wei Fan
and Qiong Chen
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139-144
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MALDI-TOF MS
spectral variation is observed between fungal samples grown under identical conditions after long-term storage by cryopreservation, freeze-drying, and under oil
Michael A. Reeve, Helen Stewart and Matthew J. Ryan
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145-151
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Effect
of melatonin on cryopreserved sperm of Prochilodus lineatus (Characiformes)
Isadora de Lima Assis, Priscila Cotta Palhares,
Gilmara Junqueira Machado, José Gilmar da Silva Souza,
Thales de Souza França, Viviane de Oliveira Felizardo
and Luis David Solis Murgas
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152-158
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Effect of EDTA
as chelating agent in extender on the post thaw quality of buffalo bull spermatozoa
Nadir Hussain, Syed Murtaza Hassan Andrabi
and Muhammad Usman Mehmood
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159-163
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Numerical
study and experimental verification of tissue cryofreezing based on flexible cryoprobe system
Tao Song, Baolin Liu, Binkai Xu and Chi Yang
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164-172
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Effect
of cryopreservation on CASA characteristics, mitochondrial transmembrane potential, plasma and acrosome integrities, morphology and in vivo fertility of buffalo bull spermatozoa
Hussain Ahmed, Sayed Murtaza Hassan Andrabi,
Syed Aftab Hussain Shah, and Sarwat Jahan
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173-180
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A
simple and reliable cooling approach for the cryopreservation of hematopoietic stem cells: the passive cooling rate-controlled technique
Yu Huang, S. M. Chapal Hossain, Kashan Memon,
Ji Peng, Jianye Wang, Zhiquan Shu, Kui Ma, Lingxiao Shen,
Frank Liu Gao, Yunxia Cao and Gang Zhao
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181-186
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Human gametes
cryopreservation with cryoprotectant modified by egg yolk
A. Grigorieva, E. Simonenko, S. Garmaeva, A. Mironova
and S. Yakovenko
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187-192
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Impact of
distinct freezing curves on the quality of ram sperm after thawing
Carlos Eduardo Ranquetat Ferreira, Karina Lemos Goularte,
Geórgia da Cruz Tavares, Stella Mari Meneghello Gheller,
Rafael Gianella Mondadori, Arnaldo Diniz Vieira
and Thomaz Lucia Jr.
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193-199
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CryoLetters 40 (3), 139-144 (2019)
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HYALURONIDASE PRETREATMENT IMPROVES THE CRYOPRESERVATION OF HUMAN OVARIAN TISSUE
Zhun Xiao1,#, Zhong-Ying Huang1,#, Yao-Yao Zhang1,
Wei Fan1, Qiong Chen2,*
1 Reproductive Medical Center of West China 2nd University Hospital, Key Laboratory
of Birth Defects and Related Diseases of women and Children, Ministry of Education, Sichuan University, Chengdu 610041, China.
2 Neonatal Intensive Care Unit of West China 2nd Hospital, Sichuan University,
Chengdu 610041, China.
# Zhun Xiao and Zhong-Ying Huang contributed equally to this work and are co-first authors
*Corresponding author: chenqiong8198@163.com
Abstract
BACKGROUND: Slow freezing has been widely applied to preserve human ovarian tissue. OBJECTIVE:
The aim of this study is to determine whether human ovarian cortex pretreated with hyaluronidase can improve the protective effect of cryopreservation. MATERIALS AND METHODS:
Human ovarian biopsies from seven patients were cryopreserved using slow freezing. The samples in the experimental group were incubated in culture medium with hyaluronidase for a short time before
cryopreservation. The histology of the thawed ovarian tissue was investigated; the levels of steroid hormone in the culture media were then used to evaluate the
development and function of thawed ovarian tissue. RESULTS: The result showed that the percentage of morphologically abnormal primordial follicles was higher in the
freezing control than in the experimental group (P<0.05). 17β-estradiol (E2) and progesterone (P4) concentrations were significantly higher in the experimental group
than in the freezing control group (P<0.05). CONCLUSION: Our study showed that human ovarian cortex pretreatment with hyaluronidase can improve the protective effect
of cryopreservation by improving the penetration of cryoprotectant.
Keywords: human ovarian tissue, slow freezing, primordial follicle, hyaluronidase, steroid hormone.
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CryoLetters 40 (3), 145-151 (2019)
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MALDI-TOF MS SPECTRAL VARIATION IS OBSERVED BETWEEN FUNGAL SAMPLES GROWN UNDER IDENTICAL CONDITIONS AFTER LONG-TERM
STORAGE BY CRYOPRESERVATION, FREEZE-DRYING, AND UNDER OIL
Michael A. Reeve*, Helen Stewart, and Matthew J. Ryan
CABI, Bakeham Lane, Egham, Surrey, TW20 9TY, UK
*Corresponding author email: M.Reeve@cabi.org
Abstract
BACKGROUND: Matrix-assisted laser-desorption and ionization time-of-flight mass
spectrometry (MALDI-TOF MS) offers a powerful, versatile, and relatively-inexpensive tool for the characterization and/or identification of protein-containing samples. OBJECTIVE:
In the case of filamentous fungi, significant variation in MALDI-TOF MS spectra can be observed for growth under different conditions on agar plates, as well
as over time for growth on the same medium. We therefore sought to investigate the possibility of additional variance as a result of the preservation conditions prior to culturing.
MATERIALS AND METHODS: We investigated Fusarium nygamai IMI 383003, previously preserved by liquid nitrogen cryopreservation, freeze-drying, and storage under oil. RESULTS:
Significant spectral differences are observed as a function of preservation conditions prior to culturing on agar plates, under experimental
conditions that show high reproducibility between replicate sample preparations and between replicate agar plates used for culturing. CONCLUSION: Storage and
cryopreservation conditions can affect MALDI-TOF MS spectra.
Keywords: fungal adaptation, freeze-drying, growth conditions, preservation conditions.
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CryoLetters 40 (3), 152-158 (2019)
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EFFECT OF MELATONIN ON CRYOPRESERVED SPERM OF Prochilodus lineatus (CHARACIFORMES)
Isadora de Lima Assisaa, Priscila Cotta Palharesaa,
Gilmara Junqueira Machadoaa, José Gilmar da Silva Souzabb,
Thales de Souza Françabb, Viviane de Oliveira Felizardobb,
Luis David Solis Murgasaa*
aDepartment of Veterinary Medicine, Federal University of Lavras, Mailbox: 3037, Lavras, Minas Gerais 37200-000, Brazil.
bDepartment of Animal Production, Federal University of Lavras, Mailbox: 3037,
Lavras, Minas Gerais 37200-000, Brazil.
*Corresponding author email: lsmurgas@dmv.ufla.br
Abstract
BACKGROUND: Semen cryopreservation is a technique widely used in commercial
and conservation fish farms. However, the success of the technique will depend on the diluent and cryoprotectant solutions added to the semen prior to the freezing procedure. OBJECTIVE:
To evaluate the effects of different doses of melatonin on motility, morphology and fertilization capacity of cryopreserved semen of Prochilodus lineatus. MATERIALS AND METHODS:
Semen was diluted in cryoprotectant solution with added melatonin, resulting in three treatments. A Control Solution (DMSO8% +
BTS5%), was compared with T1, T2 and T3 treatments: BTS (5%) + DMSO (8%) plus concentrations of 1, 2 and 3 mM of melatonin, respectively. The diluted semen was
frozen in a cylinder of vapour nitrogen and stored at - 190°C. The straws were thawed in a water bath at 40°C for 12 s for analysis of sperm motility and morphology, and for
evaluation of fertilization rate. The data were submitted to ANOVA, and the means were compared by the Tukey test. RESULTS: There was no statistical difference
(P>0.05) between the progressive motility rates and the velocities of the cryopreserved semen in the different treatments. The total motility rate was higher (P <0.05) in the
groups T1 and T2 when compared to T3, but did not differ from control. All treatments had similar values of total anomalies after thawing and did not differ in fertilization capacity (P>0.05).
CONCLUSION: The addition of melatonin on the cryoprotectant solution did not promote changes on motility, morphology and fertilization capacity of cryopreserved semen of Prochilodus lineatus.
Keywords: freezing, seminal quality, sperm class analyzer, Curimba.
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CryoLetters 40 (3), 159-163 (2019)
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EFFECT OF EDTA AS CHELATING AGENT IN EXTENDER ON THE POST THAW QUALITY OF BUFFALO BULL SPERMATOZOA
Nadir Hussain1, Syed Murtaza Hassan Andrabi2
and Muhammad Usman Mehmood1*
1 Department of Theriogenology, Faculty of Veterinary Sciences, University of
Veterinary and Animal Sciences, Lahore, Pakistan.
2 Animal Reproduction Laboratory, Animal Sciences Institute, National Agricultural Research Center, Islamabad, Pakistan.
*Corresponding author email: usman.mehmood@uvas.edu.pk
Abstract
BACKGROUND: Spermatozoa are prone to mechanical and biochemical stresses
upon freezing. The influx of Ca++ causes early capacitation and production of reactive oxygen species. OBJECTIVE: To evaluate the effect of ethylenediaminetetraacetic
acid (EDTA) as a calcium chelator in a semen extender. MATERIALS AND METHODS: The effect of EDTA concentrations (0%, 0.1%, 0.2% and 0.3%) on the
post thaw quality of buffalo bull spermatozoa were studied. RESULTS: The extender with 0.2% EDTA improved significantly visual motility, progressive motility and plasma
membrane integrity. Sperm kinematics, such as beat cross frequency (BCF), curved line velocity (VCL), straight line velocity (VSL) and average path velocity (VAP), were
higher in the extender with 0.2% EDTA. EDTA at 0.2% improves semen parameters (visual motility, supra vital plasma membrane integrity, chromatin integrity and
percentage viable spermatozoa with intact acrosome. CONCLUSION: The EDTA supplementation in a semen extender improves the post-thaw quality of Nili-Ravi buffalo bull semen.
Keywords: cryopreservation, ethylenediaminetetraacetic acid (EDTA), spermatozoa, buffalo.
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CryoLetters 40 (3), 164-172 (2019)
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NUMERICAL STUDY AND EXPERIMENTAL VERIFICATION OF TISSUE CRYOFREEZING BASED ON FLEXIBLE CRYOPROBE SYSTEM
Tao Song1, Baolin Liu1*, Binkai Xu2 and Chi Yang2
1 Institute of Biothermal Technology, University of Shanghai for Science and Technology, Shanghai, China;
2 AccuTarget MediPharma (Shanghai) Co. Ltd, Shanghai, China.
Corresponding authoræFblliuk@163.com.
Abstract
BACKGROUND: Cryosurgery is effectively used for treating various diseases including
cancer and tumor. But there is difficulty accurately quantifying the spatial temperature profile needed to determine diseased tissue destruction. OBJECTIVE: A novel flexible
cryosurgical probe was developed that can pass through the natural cavities of human body to minimize the damage to normal tissues. The present study aims to study the
design of the flexible cryoprobe by 3D heat transfer modeling and experimental verification. MATERIALS AND METHODS: A 3D model, on the basis of the flexible
cryoprobe system, was developed to predict the temperature distribution during cryosurgery in porcine liver. In vitro experiments and pathological analysis with TUNEL
staining of tissue samples were performed for verification. RESULTS: The results of numerical simulation based on the Pennes equation exhibited good agreement with
experimental measurements for the flexible cryoprobe system. The simulation shows that the tissue within the range of 0 to 6 mm along the cryoprobe is effectively
destroyed and the temperature in this domain decreases to the range -35°C to -40°C. Due to the non-uniform distribution, the temperature in the z-axis direction decreases to -40°
C faster, with more serious tissue damage; whereas the x-axis direction has the greater distance of tissue destruction. Pathologic analysis of tissue after in vitro
cryosurgery has verified the efficacy of the flexible cryoprobe system. CONCLUSION: Numerical simulation and experimental verification demonstrate the efficacy of the
flexible cryoprobe system. The 3D heat transfer model can be a valuable tool for optimizing the design of the flexible cryoprobe system.
Keywords: cryosurgery, heat transfer, numerical modelling.
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CryoLetters 40 (3), 173-180 (2019)
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EFFECT OF CRYOPRESERVATION ON CASA CHARACTERISTICS, MITOCHONDRIAL TRANSMEMBRANE POTENTIAL, PLASMA AND ACROSOME
INTEGRITIES, MORPHOLOGY AND IN VIVO FERTILITY OF BUFFALO BULL SPERMATOZOA
Hussain Ahmed1*, Sayed Murtaza Hassan Andrabi2,
Syed Aftab Hussain Shah2,3, Sarwat Jahan4
1 Department of Zoology, Women University Swabi, Khyber Pakhtunkhwa (KPK),
Pakistan
2 Animal Reproduction Laboratory, Animal Sciences Institute, National Agricultural
Research Center, Islamabad 45500, Pakistan
3 PASTIC Quaid–i–Azam University, Islamabad, Pakistan
4 Reproductive Physiology Laboratory, Department of Animal Sciences,
Quaid–i–Azam University, Islamabad, Pakistan
*Corresponding author email: ahmedmm316@gmail.com
Abstract
BACKGROUND: Application of frozen-thawed semen is an important tool for improving
the vivo fertility, but the process of freezing and thawing causes significant damage to spermatozoa. OBJECTIVE: The aim of this study was to evaluate the effect of
cryopreservation on CASA characteristics, mitochondrial transmembrane potential, plasma, and acrosome integrities, morphology and in vivo fertility of buffalo bull spermatozoa. MATERIALS AND METHODS:
Semen was collected from four mature buffalo bulls with artificial vagina at 42 °C. Ejaculates having > 1 mL volume, > 60 % sperm visual motility and > 0.5 x 109 sperm/mL concentration from each bull were
diluted in Tris–citric acid egg yolk glycerol extender (TCA) making two aliquots per bull for analysis at post dilution and cryopreserved respectively. RESULTS: Analysis of
variance (ANOVA) showed that the process of freezing and thawing significantly reduced (P < 0.05) CASA characteristics including total motility (TM, %), progressive
motility (PM, %), rapid velocity (RV, %), average path velocity (VAP, µm/sec), straight line velocity (VSL, µm/sec), curvilinear velocity (VCL, µm/sec), beat cross frequency
(BCF, Hz), straightness (STR, %) and linearity (LIN, %). Furthermore, the process of freezing and thawing significantly reduced (P < 0.05) subjective motility (SM,%),
Supra-vital plasma membrane integrity (SVPMI, %), high mitochondrial membrane potential (HMMP, %), viable spermatozoa with intact acrosome (V/IACR, %).
Moreover, it was observed that the freezing thawing process significantly decreased the in vivo fertility (%, 50.35% vs. 61.39; P < 0.05) as compared to post diluted semen. CONCLUSION
: It is concluded that the process of freezing and thawing significantly reduced semen quality and in vivo fertility of buffalo bull in terms of various functional parameters.
Keywords: cryopreservation, buffalo sperm, CASA, mitochondrial transmembrane potential.
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CryoLetters 40 (3), 181-186 (2019)
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A SIMPLE AND RELIABLE COOLING APPROACH FOR THE CRYOPRESERVATION OF HEMATOPOIETIC STEM CELLS: THE PASSIVE
COOLING RATE-CONTROLLED TECHNIQUE
Yu Huang1, 2, S. M. Chapal Hossain1, Kashan Memon1, Ji Peng3,
Jianye Wang2, 4, Zhiquan Shu3, 5, Kui Ma6, Lingxiao Shen1, 2,
Frank Liu Gao7, Yunxia Cao2, 4, Gang Zhao1, 2*
1 Centre for Biomedical Engineering, Department of Electronic Science and
Technology, University of Science and Technology of China, Hefei, China;
2 Anhui Provincial Engineering Technology Research Center for Biopreservation and Artificial Organs, Hefei, China;
3 Department of Mechanical Engineering, University of Washington, Seattle, WA 98195, USA;
4 Center for Reproductive Medicine, Obstetrics and Gynecology, The First Affiliated
Hospital of Anhui Medical University, Hefei, China;
5 School of Mechanical and Materials Engineering, Washington State University, Everett, WA 98201, USA;
6 Hebei Life Origin Bio-Technology Co., Shijiazhuang, China;
7 School of Public Affairs, University of Science and Technology of China, Hefei, China.
*Corresponding author: zhaog@ustc.edu.cn
Abstract
BACKGROUND: Cryopreservation of hematopoietic stem cells (HSCs) has widely
been used in stem cell transplantation and cellular therapy treating various human diseases. However, the current conventional cooling approach for the cryopreservation
of HSCs has the following potential problems: (1) requirement of a very expensive computer-programmed liquid nitrogen freezer (LNF) for the cooling rate control, (2) a
large consumption of liquid nitrogen, (3) periodic breakdown of the LNF due to the mechanical failure of the liquid nitrogen valves (i.e., magnetic- solenoid valves) inside
the LNF, and (4) constant monitoring of the LNF operation during the HSCs cooling process. OBJECTIVE: To test and evaluate a simple and reliable approach for the
cryopreservation of HSCs using the passive cooling technique. MATERIALS AND METHODS: A passive cooling-rate-controlled device (PCD) was developed and used to
cryopreserve HSCs. The PCD is inexpensive, simple, and user-friendly, which needs only the minimum maintenance and no consumption of liquid nitrogen. The PCD was
compared to the LNF for the cryopreservation of HSCs in the present study through experiments. The cell viability and functionality were evaluated after cryopreservation.
RESULTS: In comparison with the LNF method, the PCD approach enabled high cell viability/survival, recovery rate, and functionality after cryopreservation processes. CONCLUSION:
The PCD offers a cost- effective, simple, and reliable approach for the optimal cryopreservation of HSCs.
Keywords: cryopreservation, hematopoietic stem cells, passive cooling rate-controlled
device.
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CryoLetters 40 (3), 187-192 (2019)
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HUMAN GAMETES CRYOPRESERVATION WITH CRYOPROTECTANT MODIFIED BY EGG YOLK
A. Grigorievaa, E. Simonenkoa*, S. Garmaevaa,
A. Mironovab, S. Yakovenkoa
a Lomonosov Moscow State University, Physical Department, GSP-1, Leninskie gory, Moscow, Russia, 119991;
b Semenov Institute of Chemical Physics, 4 Kosygina street, building 1, Moscow,
Russia, 119991
Corresponding author email: ksimonenko@inbox.ru
Abstract
BACKGROUND: The modification of cryoprotectants with an egg yolk is an effective method to improve cell survival. OBJECTIVE:
This study is dedicated to the comparison of four variants of cryoprotectants modified with egg yolk (commercially available and custom developed with yolk emulsion at 5.6 mg ml-1) and with lecithin.
METHODS: Cryoprotectant effectiveness was evaluated by cytotoxicity and vital screen tests, and by the determination of sperm motility index. RESULTS: A simple
analytical model has been created to determine an approximate concentration of cholesterol in the solution sufficient to stabilize cellular membranes. It was shown that
the average fractions of living cells, motility and membrane integrity were higher for our modification. CONCLUSION: We conclude that phosphatidylcholine (1.5 mg ml-1)
contributes to the cryoprotective action of egg yolk, but doesn`t define it entirely. The cryoprotective effect of egg yolk on plasma membranes is complex, and isn`t caused
by the action of one of its components.
Keywords: cryopreservation, egg yolk, sperm quality, fertilization, model of cholesterol concentration.
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CryoLetters 40 (3), 193-199 (2019)
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I
MPACT OF DISTINCT FREEZING CURVES ON THE QUALITY OF RAM SPERM AFTER THAWING
Carlos Eduardo Ranquetat Ferreira1,2, Karina Lemos Goularte1,2,
Geórgia da Cruz Tavares1,2, Stella Mari Meneghello Gheller1,2,
Rafael Gianella Mondadori1,3, Arnaldo Diniz Vieira1,2,
Thomaz Lucia Jr.1,2*
1 ReproPel,
2 Faculdade de Veterinária,
3 Instituto de Biologia Universidade Federal de Pelotas, 96010-900, Pelotas-RS, Brazil
*Corresponding author email: thomaz@ufpel.edu.br, thomaz@pq.cnpq.br
Abstract
BACKGROUND: Automated equipment with customized freezing curves can be used
to cryopreserve ram sperm, but none is considered a standard. OBJECTIVE: This study compared the post-thawing quality of ram sperm frozen using a conventional
freezing curve and two controlled-rate curves. MATERIALS AND METHODS: Six ejaculates were collected from four rams (n = 24). In the conventional curve (110 min),
sperm was cooled at -0.3 to - 0.5°C min-1 until 5°C, stabilized for 60 min and exposed to liquid nitrogen (LN2) vapor for 10 min (-80°C) before submersion. The
slow-customized (SC) curve (126.2 min) used a rate of - 0.25°C min-1 until 5°C, stabilization for 60 min and a rate of -20°C min-1 until -120°C before immersion in LN2.
Rates for the fast-customized (FC) curve (75 min) were: -0.3°C min-1 until 5°C; -3°C min-1 until -10°C; -5°C min-1 until -35°C; and -4°C min-1 until immersion in LN2 (- 43°C).
RESULTS: Velocity in a straight line and beat-cross frequency were lower for spermatozoa frozen with the FC than with the conventional curve (P < 0.05). The FC
curve resulted in more membrane and acrosome damages than the other curves (P < 0.05). Mitochondrial membrane potential was lower with the SC than with the other curves (P < 0.05).
CONCLUSION: The conventional curve was more efficient than both tested automated freezing curves. The FC curve may be an alternative to the SC curve due to the shorter processing time.
Keywords: freezing, controlled-rate, membrane, acrosome, sperm kinetics, ram.
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