Cryopreservation of saltwater crocodile (Crocodylus porosus) spermatozoa

2017 ◽  
Vol 29 (11) ◽  
pp. 2235 ◽  
Author(s):  
S. D. Johnston ◽  
E. Qualischefski ◽  
J. Cooper ◽  
R. McLeod ◽  
J. Lever ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Membrane Integrity ◽  
Sperm Cells ◽  
Plasma Membrane Integrity ◽  
Final Study ◽  
Combined Use ◽  
Saltwater Crocodile ◽  
Crocodylus Porosus ◽  
Phosphate Buffered Saline

The aim of the present study was to develop a protocol for the successful cryopreservation of Saltwater crocodile spermatozoa. Sperm cells were frozen above liquid nitrogen vapour in phosphate-buffered saline (PBS) containing either 0.3 M trehalose, 0.3 M raffinose or 0.3 M sucrose and compared with glycerol (0.3–2.7 M). Although the highest levels of mean post-thaw motility were observed following cryopreservation in 0.3 M trehalose (7.6%) and 0.3 M sucrose (7.3%), plasma membrane integrity (PI) was best following cryopreservation in 2.7 M glycerol (52.5%). A pilot study then assessed the cytotoxicity of glycerol and sucrose prior to cryopreservation and revealed no loss of survival when spermatozoa were diluted in 0.68 M glycerol or 0.2–0.3 M sucrose once cryoprotectants were washed out with PBS or Biggers, Whitten and Whittingham medium containing sperm capacitation agents (BWWCAP). A final study refined the combined use of permeating (0.68 or 1.35 M glycerol) and non-permeating (0.2 or 0.3 M sucrose) cryoprotectants. Spermatozoa were cryopreserved in liquid nitrogen vapour at rates of approximately −21°C min−1 (fast freeze) or −6.0°C min−1 (slow freeze). Post-thaw survival was highest with a combination of 0.2 M sucrose and 0.68 M glycerol and when these cryoprotectants were washed out with BWWCAP, regardless of whether spermatozoa were frozen using a fast (motility 14.2 ± 4.7%; PI 20.7 ± 2.0%) or slow (motility 12.0 ± 2.7%; PI 22 ± 4%) cryopreservation rate.

217 QUALITY CHANGES IN POST-THAW SPRINGBOK (ANTIDORCAS MARSUPIALIS) EPIDIDYMAL SPERM MAINTAINED IN FERTILIZATION MEDIUM

2006 ◽  
Vol 18 (2) ◽  
pp. 216
Author(s):  
F. P. Chatiza ◽  
G. M. Pieterse ◽  
P. Bartels
Keyword(s):  
South Africa ◽  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Membrane Integrity ◽  
Water Bath ◽  
Quality Changes ◽  
Epididymal Sperm ◽  
Plasma Membrane Integrity ◽  
Free Ranging ◽  
Over Time

The availability of gametes from the cropping of excess wildlife species provides the opportunity for the advancement of knowledge into assisted reproductive technology for possible future conservation measures. Little is known about the longevity of springbok (Antidorcas marsupialis) spermatozoa maintained in fertilization medium. The aim of this project was to determine the quality changes of post-thawed springbok spermatozoa incubated in fertilization medium by measuring plasma membrane integrity over time. Testes (n = 12) were obtained from two geographically distinct free-ranging springbok populations in South Africa. Spermatozoa were flushed from the cauda epididymides within three hours of the animals' death. Samples from an individual male were pooled, diluted to 400 × 106 sperm/mL with Biladyl (Minitüb, Tiefenbach, Germany) fraction A (no glycerol) and equilibrated in a water bath for 6 h at 4°C. An equal volume of Biladyl fraction B (containing 12% glycerol) was added to the sample to make a final concentration of 200 × 106 sperm/mL. Samples were loaded into 0.25-mL straws and frozen in liquid nitrogen vapor (5 cm above the liquid nitrogen level) for 20 min after which they were plunged into liquid nitrogen. Straws from each sample were thawed for 20 s at 36°C in a water bath. Thawed spermatozoa (100 μL) was added to 1 mL IVF-TALP medium containing heparin and PHE (Vajta et al. 1996 Theriogenology 45, 683–689) in 2-mL Nunc tubes (AEC, Amersham, South Africa) and incubated at 38.7°C, in humidified 5% CO2 balance air for 30 h. Aliquots were extracted from the incubating spermatozoa to determine plasma membrane integrity at 6-h intervals. Propidium iodide (Sigma, South Africa) at 50 ng/mL (10 min at RT) was used to evaluate membrane integrity under fluorescence microscopy at ×400, with a 450-nm excitation filter, a 510-nm dicroic beam splitter, and a 520-nm barrier filter. Cells with damaged plasma membrane have nuclei that fluorescence red. Eosin/nigrosin was also used to evaluate membrane integrity under ×400 bright-field microscopy. Cells with damaged plasma membrane stain purple-red, whereas the balance of cells remain translucent. The average post-thaw motility of spermatozoa in populations A and B was 69% (n = 6) and 68% (n = 6), respectively. Plasma membrane integrity of post-thawed springbok spermatozoa deceased steadily in IVF-TALP medium over the 30-h period (Table 1). Cryopreserved epididymal sperm derived from free-ranging springbok populations survive in IVF-TALP media and may be useful in future conservation activities where an isolated gene pool requires genetic supplementation through one or more assisted reproduction techniques such as IVF or AI. Further research is required to confirm and extend these findings. Table 1. Percentage plasma membrane integrity of post-thawed springbok sperm over time

20 LIPID PEROXIDATION AND GENERATION OF HYDROGEN PEROXIDE FROM SUBFERTILE STALLION SPERMATOZOA DURING STORAGE AT REFRIGERATION TEMPERATURE

2013 ◽  
Vol 25 (1) ◽  
pp. 157 ◽  
Author(s):  
P. N. Guasti ◽  
C. P. Freitas-Dell'aqua ◽  
R. R. D. Maziero ◽  
G. A. Monteiro ◽  
F. P. Hartwig ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Plasma Membrane ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Membrane Lipid ◽  
Sperm Cells ◽  
Band Pass Filter ◽  
Membrane Lipid Peroxidation ◽  
Pass Filter ◽  
Plasma Membrane Integrity

The aim of this study was to evaluate the generation of hydrogen peroxide (H2O2) and membrane lipid peroxidation of subfertile spermatozoa stored at 5°C for 24 h. Semen samples, collected from 5 subfertile stallions (≤40% of conception rate), were diluted in a skim-based extender (BotuSemen; Botupharma) and stored in a passive transport container at 5°C over a period of 24 h. Sperm motility was determined by computer-assisted semen analysis (CASA; IVOS 12, Hamilton Thorne Inc., Beverly, MA, USA) for total motility (TM), progressive motility (PM), and rapid sperm (RAP). Plasma membrane integrity, generation of H2O2, and membrane lipid peroxidation were determined by flow cytometry (LSRFortessa cell analyzer, BD Biosciences, Franklin Lakes, NJ, USA). For evaluation of acrosome and plasma membrane integrity, samples were stained with Hoechst 33342 dye (H33342; Molecular Probes, Eugene, OR, USA), iodide propidium (IP; Sigma, St. Louis, MO, USA), and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA; Sigma). For the assessment of generation of H2O2, 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma), H33342, and IP were added to the sperm suspension. For the assessment of sperm lipid peroxidation, samples were stained with C11-BODIPY581/591 (Molecular Probes), H33342, and IP. Samples were evaluated after semen collection (0 h) and after 24 h of cooled storage. A total of 10 000 gated events were analyzed per sample by flow cytometry. The green fluorescence (FL1) was collected through a 580-nm band-pass filter and the red fluorescence (FL3) through a 635-nm band-pass filter. Statistical analysis was performed using GraphPad Prism version 4.03 (2005; GraphPad Software Inc., La Jolla, CA, USA), through paired t-test to identify the significant differences (P ≤ 0.05). In general, sperm motility parameters of TM and RAP, and acrosome and plasma membrane integrity significantly decreased after 24 h of refrigeration at 5°C (P ≤ 0.05). Interestingly, no differences were found in PM at 0 and 24 h (P ≥ 0.05). The percentage of sperm cells with high generation of H2O2 did not differ at 0 and 24 h (P ≥ 0.05), whereas the percentage of sperm cells with membrane peroxidation increased (0.56 ± 0.3 v. 2.24 ± 1.3; P ≤ 0.05) at these periods. The percentage of viable sperm cells with low generation of H2O2 had a significant decrease from 22.6 ± 11.2 at 0 h to 0.08 ± 0.1 after 24 h of storage (P ≤ 0.05), although no differences were found in the percentage of viable sperm cells without membrane peroxidation (P ≥ 0.05). In conclusion, the cooled storage of subfertile spermatozoa for 24 h drastically decreased the number of viable spermatozoa with low generation of H2O2 and increased the percentage of membrane lipid peroxidation, which is related to the decrease in sperm motility and increase in dead sperm. These results make it difficult to use refrigerated semen of subfertile stallions with poor semen quality in commercial breeding programs. São Paulo Research Foundation (FAPESP) is acknowledged for supporting this research.

12 FERTILITY OF FROZEN EQUINE SPERM IN SYSTEMS FOR CRYOPRESERVATION

2012 ◽  
Vol 24 (1) ◽  
pp. 117
Author(s):  
R. R. D. Maziero ◽  
P. N. Guasti ◽  
I. D. P. Blanco ◽  
I. Martin ◽  
G. A. Monteiro ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Membrane Integrity ◽  
Skim Milk ◽  
Egg Yolk ◽  
Automated System ◽  
Epifluorescence Microscopy ◽  
Computer Assisted ◽  
Plasma Membrane Integrity ◽  
Sperm Analysis

Optimizing cryopreservation of equine sperm will facilitate genetic banking and propagation of important horse strains through assisted reproduction. This study aimed to evaluate the motility pattern using computer-assisted sperm analysis (CASA) and plasma membrane integrity by epifluorescence microscopy of equine semen frozen in 0.5 mL straws at different freezing rates; also, a fertility trial was performed according to the freezing protocol. Three ejaculates from four stallions of various breeds (Mangalarga Marchador, Westfallen, Hanovarian and Arabian) and ages (5 to 20 years) were collected and processed for cryopreservation. The stallions were housed at the CERBEQ, Reproduction Centre of the Department of Animal Reproduction and Veterinary Radiology, UNESP. The ejaculates were filtered and submitted to analysis by CASA (HTM IVOS 12, Hamilton Thorne Research, USA). In addition, the plasma membrane integrity was determined by fluorescent probes. After evaluation, the ejaculates were diluted at 1:1 (extender:semen) with skim milk extender Botu-Semen™ and centrifuged at 600 × g for 10 min. The supernatant was removed and the pellet resuspended to a final concentration of 100 × 106 sperm mL–1 with milk-egg yolk freezing extender (Botu-Crio™). Semen was packaged in 0.5-mL straws (IMV, LAigle, France) and was placed in nitrogen for 20 min and then from room temperature to 5°C and then frozen in two different cooling systems: an isothermic box (42 cm × 28 cm × 12.5 cm) was placed upon racks suspended 6 cm above liquid nitrogen or other 20 min then immersed into nitrogen and automated system Mini Digitcool™ (IMV Technologies, France), cooling at a –40°C min–1 rate. All straws were stored in liquid nitrogen until thawing and analysis. The straws were thawed in a water bath at 46°C for 20 s and the samples were evaluated for progressive motility, angular progressive velocity, progressive velocity, track speed, percentage of rapid sperm and percentage of sperm with plasma membrane integrity. For the fertility trial, 65 clinically healthy mares had their oestrous cycle monitored by ultrasound and inseminated postovulation with sperm into the uterus. Ovulation was induced with 1 mL of deslorelin acetate (GnRH) injected IM when a 35-mm follicle was detected. Thirty-six hours later, mares were monitored every 6 h until ovulation was detected. When it was detected, mares were inseminated with 800 × 106 total sperm. Pregnancy was confirmed via ultrasound examination 15 days after ovulation. Pregnancy rate was 52.2% using the isothermic box and 60% using the automated machine. Statistical analysis from the frozen–thawed semen evaluated parameters was performed using the statistics software Proc. MIXED of SAS 9.1 and for the fertility trial, logistic regression using the Proc GENMOD from SAS 9.1. The conventional method using the isothermic box was similar to the automated machine with a fast freezing rate. Additionally, AI with 800 × 106 sperm frozen in the isothermic box or automated system resulted in similarly acceptable conception rates.

Factors affecting storage of Slovak native rabbit semen in the gene bank

Zygote ◽  
2017 ◽  
Vol 25 (5) ◽  
pp. 592-600
Author(s):  
Barbora Kulíková ◽  
Marta Oravcová ◽  
Andrej Baláži ◽  
Peter Supuka ◽  
Peter Chrenek
Keyword(s):  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Semen Quality ◽  
Sperm Quality ◽  
Objective Assessment ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Quality Traits ◽  
Plasma Membrane Integrity

SummaryIn this study, fresh and frozen–thawed semen of Nitra and Zobor rabbit breeds were evaluated for potential inter-breed or inter-male differences in sperm quality traits. Individual male semen from four rabbits of each breed were diluted (v:v; 1:1) in a freezing medium composed of a commercial diluent, 16% of dimethyl sulphoxide (DMSO), 4% of Ficoll 70 and 2% of sucrose and frozen in liquid nitrogen vapours before being plunged into liquid nitrogen. Different motility traits, viability and plasma membrane integrity of fresh and frozen–thawed semen were evaluated in vitro using computer-assisted sperm analysis and flow cytometry. To evaluate the sperm fertilization ability, artificial insemination of fresh and frozen–thawed sperm was performed. Our results showed the effect of breed (P ≤ 0.05) on frozen–thawed sperm viability and plasma membrane integrity. Moreover, individual variability in semen quality among the rabbits was revealed (0.31 to 0.71 among quality traits). Our results thereby confirmed that the cryopreservation procedure could not ensure comparable sperm post-thaw survival for different breeds or males. Nevertheless, correlations between numbers of fresh total motile and progressively moving sperm and several quality parameters measured post thawing were revealed. Therefore, we suggest that the objective assessment of fresh rabbit sperm motility may be an effective indicator of frozen–thawed semen quality. Consequently, regular semen assessment is required in order to preserve good-quality insemination doses from native breeds.

scholarly journals Influence of Coconut Powder Water-Based Conservation Medium (APC-102c) for Maintaining Mitochondrial Activity of Cryopreserved Ram Sperm

2019 ◽  
Vol 47 (1) ◽  
Author(s):  
Bruna Farias Brito ◽  
Bárbara Mara Bandeira Santos ◽  
Leonardo Alves Rodrigues Cabral ◽  
David Baruc Cruvinel Lima ◽  
Cristiane Clemente De Melo Salgueiro ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Artificial Insemination ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Mitochondrial Activity ◽  
Plasma Membrane Integrity ◽  
Significant Difference ◽  
Motility Parameters

Background:  Semen extenders are required to protect and preserve semen, and the development of suitable extenders is key for artificial insemination. Although the use of Tris-based diluent is widespread, new diluents such as powdered coconut water have been developed for better sperm protection. One way to evaluate the effectiveness of diluents is through microscopic analyses that evaluate sperm motility, vigor, and concentration. However, these analyses are limited, and may not provide accurate results. New evaluation techniques have been studied, and one of the tests that can be used to add reliability to these analyses is mitochondrial activity evaluation, which can sum all the parameters, and provide a more accurate evaluation. Thus, the present study aimed to evaluate the efficacy of ACP-102c in cryopreserved ram semen.Materials, Methods & Results: Five semen samples were collected from two ram breeders using artificial vagina (n = 10). Each ejaculate was divided into the following two treatments: T1 - ACP-102c + 20% egg yolk + 7% glycerol and T2 - TRIS + 20% egg yolk + 7% glycerol. Extended semen samples were then packed in 0.5 mL plastic straws, subjected through the refrigeration curve up to 4°C (0.35° C/min), and equilibrated for 2 h at 4°C. Subsequently, the straws were placed at 4 cm above liquid nitrogen level (-60°C) for 15 min, immersed, and then finally stored in the liquid nitrogen at -196°C. Both fresh and thawed samples were evaluated for total and progressive sperm motility using conventional microscopy (40x), and the same evaluator on each occasion. For plasma membrane integrity (IMP), the smear staining technique with the Eosin-Nigrosin staining was used; 200 sperms were counted and classified as whole (unstained) and unhealthy (stained). Mitochondrial activity was evaluated using a cytochemical technique based on the oxidation of 3,3'-diaminobenzidine (DAB); 200 sperms were counted, and classified into four classes (I, II, III, and IV) according to the degree of coloration of the intermediate part. Fresh semen showed no significant difference (P > 0.05) between treatments with respect to motility parameters; however, T2 showed significantly inferior results regarding plasma membrane integrity. After thawing, T2 was significantly higher in sperm motility parameters compared to T1. The mitochondrial activity and plasma membrane integrity parameters did not show any significant difference between the treatments.Discussion: The TRIS-based diluent showed higher motility values than ACP-102c; however, motility rates in ACP-102c diluent, although lower, are considered satisfactory for insemination, which requires semen with minimal progressive motility of 30%. Notably, the cryopreservation protocol used in this study is the standard for TRIS-based diluent, and it is known that the optimal rate of refrigeration and cryopreservation may differ according to the composition of the storage medium; therefore, we may assume that the protocol used is not yet appropriate for the ACP-102c diluent, and further studies are required. IMP is an essential attribute for fertilization, and cryopreservation can affect the plasma membrane as observed in this study. Cryopreserved semen reduced the percentage of class I mitochondrial reaction sperms in both treatments, demonstrating that cryopreservation affects the mitochondrial activity of the intermediate portion of the sperm; however, there was no difference between treatments in thawed semen. Thus, we concluded that the ACP-102c conservation medium maintains seminal quality after thawing, and it can be used in artificial insemination processes.

Comparision of DNA fragmentantion and plasma membrane integrity between chilled and frozen semen of bulls

2014 ◽  
Author(s):  
Mello Papa Patricia de ◽  
Carlos Ramires Neto ◽  
Priscilla Nascimento Guasti ◽  
Rosiara Rosaria Dias Maziero ◽  
Yame F R Sancler-Silva ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Integrity ◽  
Plasma Membrane Integrity ◽  
Frozen Semen

scholarly journals Plasma membrane integrity in health and disease: significance and therapeutic potential

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Catarina Dias ◽  
Jesper Nylandsted
Keyword(s):  
Plasma Membrane ◽  
Therapeutic Potential ◽  
Calcium Influx ◽  
Membrane Integrity ◽  
Cell Types ◽  
Physical Parameters ◽  
Membrane Repair ◽  
Plasma Membrane Integrity ◽  
Repair Mechanisms ◽  
And Function

AbstractMaintenance of plasma membrane integrity is essential for normal cell viability and function. Thus, robust membrane repair mechanisms have evolved to counteract the eminent threat of a torn plasma membrane. Different repair mechanisms and the bio-physical parameters required for efficient repair are now emerging from different research groups. However, less is known about when these mechanisms come into play. This review focuses on the existence of membrane disruptions and repair mechanisms in both physiological and pathological conditions, and across multiple cell types, albeit to different degrees. Fundamentally, irrespective of the source of membrane disruption, aberrant calcium influx is the common stimulus that activates the membrane repair response. Inadequate repair responses can tip the balance between physiology and pathology, highlighting the significance of plasma membrane integrity. For example, an over-activated repair response can promote cancer invasion, while the inability to efficiently repair membrane can drive neurodegeneration and muscular dystrophies. The interdisciplinary view explored here emphasises the widespread potential of targeting plasma membrane repair mechanisms for therapeutic purposes.

scholarly journals Plasma membrane integrity: implications for health and disease

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Dustin A. Ammendolia ◽  
William M. Bement ◽  
John H. Brumell
Keyword(s):  
Plasma Membrane ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Whole Body ◽  
Plasma Membrane Integrity ◽  
Plasma Membrane Damage ◽  
Cellular Environment ◽  
Health And Disease ◽  
Repair Pathways ◽  
The Impact

AbstractPlasma membrane integrity is essential for cellular homeostasis. In vivo, cells experience plasma membrane damage from a multitude of stressors in the extra- and intra-cellular environment. To avoid lethal consequences, cells are equipped with repair pathways to restore membrane integrity. Here, we assess plasma membrane damage and repair from a whole-body perspective. We highlight the role of tissue-specific stressors in health and disease and examine membrane repair pathways across diverse cell types. Furthermore, we outline the impact of genetic and environmental factors on plasma membrane integrity and how these contribute to disease pathogenesis in different tissues.

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