scholarly journals Importance of cooling rate and animal variability for boar sperm cryopreservation: insights from the cryomicroscope

Reproduction ◽  
2002 ◽  
pp. 315-322 ◽  
Author(s):  
A Medrano ◽  
PF Watson ◽  
WV Holt
Keyword(s):  
Plasma Membrane ◽  
Cooling Rate ◽  
Propidium Iodide ◽  
Membrane Integrity ◽  
Cooling Rates ◽  
Boar Sperm ◽  
Sperm Plasma Membrane ◽  
Thawing Process ◽  
Set Up ◽  
Two Stages

A series of experiments was set up to investigate the effect of different cooling rates on boar sperm cryosurvival using cryomicroscopy. The cooling protocols were split into two stages: (i) from +5 degrees C to -5 degrees C and (ii) from -5 degrees C to -50 degrees C. Fluorescent probes (SYBR14 and propidium iodide) were used to monitor plasma membrane integrity during the entire process. Cooling rates in the range 3 degrees C min(-1) to 12 degrees C min(-1) did not cause significant damage to the sperm plasma membrane between +5 degrees C and -5 degrees C; however, spermatozoa cooled at 24 degrees C min(-1) to -5 degrees C were slightly damaged. Motility was not particularly sensitive to variations in cooling rate. Cooling rates in the range 15 degrees C min(-1) to 60 degrees C min(-1) did not produce differences in sperm cryosurvival during freezing between -5 degrees C and -50 degrees C, or after thawing. In addition, cooling rates in the range 3 degrees C min(-1) to 80 degrees C min(-1) did not produce significant differences in sperm cryosurvival. However, slow freezing (3 degrees C min(-1)) induced a slight increase in the percentage of plasma membrane-damaged spermatozoa (propidium iodide-positive) at -50 degrees C. Inter-ejaculate and inter-boar differences in sperm cryosurvival were manifested independently of cooling rate. The sperm plasma membrane remained intact (SYBR14-positive) during cooling and freezing, but upon rewarming, the plasma membrane of a high proportion of spermatozoa was damaged (propidium iodide-positive), indicating that rewarming is a critical step of the freezing-thawing process.

Boar semen can tolerate rapid cooling rates prior to freezing

2011 ◽  
Vol 23 (5) ◽  
pp. 681 ◽  
Author(s):  
Jorge D. Juarez ◽  
Inma Parrilla ◽  
Juan M. Vazquez ◽  
Emilio A. Martinez ◽  
Jordi Roca
Keyword(s):  
Cooling Rate ◽  
Flow Cytometric Analysis ◽  
Membrane Integrity ◽  
Annexin V ◽  
Objective Evaluation ◽  
Membrane Lipid ◽  
Cooling Rates ◽  
Rapid Cooling ◽  
Motile Cells ◽  
Boar Spermatozoa

Two experiments were performed in the present study that demonstrated that boar spermatozoa are capable of surviving rapid cooling rates within a range of 15–5°C before freezing. Boar ejaculates diluted in Beltsville thawing solution (BTS) (1 : 1, v/v) were held at 17–20°C and shipped over a 24-h time period from two AI centres to a cryobiology laboratory, where they were pooled (Experiment 1) or cryopreserved individually (Experiment 2) using a standard 0.5-mL straw freezing protocol. The effects of cooling before freezing were assessed after thawing through the objective evaluation of sperm motility and flow cytometric analysis of membrane integrity, acrosomal status, changes in membrane lipid architecture monitored by merocyanine and annexin V binding and intracellular production of reactive oxygen species. In Experiment 1 (six replicates), two semen pools (five ejaculates per pool) were cooled from 15 to 5°C at rates of 0.08, 0.13, 0.40 and 1.50°C min–1. These cooling rates did not result in any significant differences (P > 0.05) in any of the post-thaw sperm assessments, even in thawed samples incubated under capacitation conditions. In Experiment 2, three individual ejaculates from 16 boars were slowly (0.08°C min–1) or rapidly (1.5°C min–1) cooled before freezing. A consistent interboar variability (P < 0.01) was detected, which was independent of the cooling rate used. Cooling rate only significantly influenced (P < 0.05) sperm assessments in four of 16 boars, which exhibited slightly higher percentages of motile cells and intact plasma and acrosomal membranes in the samples that had been cooled slowly. These findings demonstrate that boar spermatozoa undergoing cryopreservation can withstand rapid cooling rates before freezing.

scholarly journals The Relationship between Acrosome Reaction and Polyunsaturated Fatty Acid Composition in Boar Sperm

2019 ◽  
Author(s):  
Sang-Hee Lee ◽  
Yu-Jin Kim ◽  
Byeong Ho Kang ◽  
Choon-Keun Park
Keyword(s):  
Fatty Acids ◽  
Fatty Acid Composition ◽  
Plasma Membrane ◽  
Fatty Acid ◽  
Acid Composition ◽  
Acrosome Reaction ◽  
Membrane Integrity ◽  
Plasma Membrane Integrity ◽  
Boar Sperm ◽  
The Relationship

This study investigated the relationship of acrosome reactions and fatty acid composition on fertility in boar sperm. The acrosome reaction of sperm was induced via methyl-beta-cyclodextrin (MBCD), and acrosome reaction, plasma membrane integrity, and fertility were analyzed. The fatty acid composition of the excess acrosome reacted sperm was determined via gas chromatography. The results showed that the acrosome reaction in sperm was induced over 85% of the time by 60 mM MBCD treatment, and the plasma membrane integrity was significantly decreased and was dependent on the MBCD level. The acrosome reacted sperm resulted in significantly higher saturated fatty acids (SFAs) and lower unsaturated fatty acids (PUFAs) than the non-acrosome reaction group. Moreover, the acrosome reacted sperm from 60 mM MBCD significantly decreased in vitro fertility and blastocyst formation relative to non-acrosome reacted sperm, and the acrosome reaction was positively correlated with SFAs and negatively correlated with PUFAs. Of these fatty acids, C22:5n-6 (docosapentaenoic acid [DPA]) and C22:6n-3 (docosahexaenoic acid [DHA]) were directly negatively correlated with the acrosome reaction (r = -0.982 and -0.947, respectively). In conclusion, the excessive acrosome reactions may occur by reducing the PUFAs, which may then dramatically decrease sperm fertility in pigs.

scholarly journals 159 Laparoscopic insemination method in sheep allows the use of an animal protein-free and inexpensive freezing medium

2020 ◽  
Vol 32 (2) ◽  
pp. 206
Author(s):  
L. Gavin-Plagne ◽  
L. Boyer ◽  
A. Baudot ◽  
M. Guedes Teixeira ◽  
G. Louis ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Propidium Iodide ◽  
Random Effect ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Positive Control ◽  
Negative Control ◽  
Motion Parameter ◽  
Control Medium ◽  
Cooling Rates

Animal-derived products are widely used in sperm cryopreservation for their cryoprotective properties. These components, however, must be replaced because of sanitary risks. STEMALPHA.CRYO3 (Ref. 5617, Stem Alpha), called CRYO3, is a chemically defined preservation medium currently used for freezing human tissue and adult stem cells. The aim of this study was to evaluate the effects of a CRYO3-based medium and of two cooling rates on invitro parameters and invivo fertility of ram sperm. Six rams (Blanche du Massif Central) were subjected to sperm collection four times using an artificial vagina. Sperm were split and frozen in three media: an egg yolk and milk-based medium (positive control), a CRYO3-based medium (tested medium), and a medium without additives (negative control). The two cooling rates were related to the distance between the straws and the surface of liquid nitrogen during the freezing process (5 and 20cm). Sperm membrane integrity (propidium iodide/SYBR-14), acrosome integrity (fluorescein isothiocyanate-peanut agglutinin/propidium iodide; FITC-PNA/PI), and mitochondrial membrane potential (JC-1) were assessed using flow cytometry, whereas functional membrane integrity was assessed using a hypo-osmotic swelling test and motion characteristics were evaluated using computer-assisted sperm analysis. Pregnancy rate, parturition rate, and prolificacy were evaluated after performing laparoscopic inseminations (n=75 ewes). Moreover, we characterised the freezing media thermodynamically using a differential scanning calorimeter. Statistical analyses were performed using R software. Invitro parameters were assessed using a mixed model including the time and the medium as fixed effects and the ram as a random effect. Pregnancy and parturition rates, following a binomial distribution, and prolificacy, assumed to follow a Poisson distribution, were analysed using generalised linear models, including the medium as a fixed effect and the ram as a random effect. Differences with P&lt;0.05 were considered statistically significant. The cooling rates had no significant effect except on the wobble motion parameter. The positive control medium showed significantly higher results than the CRYO3-based medium and the negative control medium for all invitro parameters except for straightness motion parameter. Conversely, field trials showed no significant difference between the media for pregnancy rate (71, 64, and 74%), parturition rate (68, 61, and 74%) and prolificacy (2.0, 2.1, and 1.7), for the positive control, CRYO3-based medium, and the negative control, respectively. This study showed that the product, CRYO3, cannot replace egg yolk and milk in freezing extenders. Moreover, we showed that laparoscopic inseminations allowed a 74% parturition rate due to an easy and inexpensive medium comprising only a Tris buffer and glycerol. Although it could not be used on a large scale, this medium remains an option for international transport or long-term storage of genetic diversity.

319 VALIDATION OF THE CARBOXYFLUORESCEIN DIACETATE ASSOCIATED WITH PROPIDIUM IODIDE FOR MURINE SPERM ACROSOME AND PLASMA MEMBRANE INTEGRITY

2010 ◽  
Vol 22 (1) ◽  
pp. 315
Author(s):  
F. M. Sevciuc ◽  
C. M. Mendes ◽  
F. R. O. de Barros ◽  
W. B. Feitosa ◽  
R. Simões ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Propidium Iodide ◽  
Linear Regression Analysis ◽  
Membrane Integrity ◽  
Transgenic Animals ◽  
Exogenous Dna ◽  
Plasma Membrane Integrity ◽  
Standard Curve ◽  
Acrosomal Membrane ◽  
Simultaneous Evaluation

The spermatozoa is an ideal vehicle for genetic modification, production of transgenic animals, as well as a biotechnological tool for sperm-mediated gene transfer. However, in order to achieve successful sperm fertilization and exogenous DNA integration, it is necessary for viable cells to remain intact, allowing the sperm to penetrate the oocyte. Fluorescent probes allow evaluation of morphological and functional characteristics of cells, which can be evaluated separately or simultaneously. Therefore, the aim of this study was to validate the simultaneous evaluation of the integrity of plasma and acrosomal membranes of murine sperm using the probes carboxyfluorescein diacetate (CF) and propidium iodide (PI). In order to validate, a standard curve was performed. Sperm were obtained from epididymis and vas deferens from CD-1 mice (8 to 16 weeks of age). Recovered samples were diluted in PBS and then divided into 2 aliquots: one prepared with fresh semen (FS) and the other submitted to Percoll gradient (45%/90%) followed by flash-freezing in liquid nitrogen and thawing (FTP) to induce acrosome damage. Samples were prepared with the following average of FS:FTP: 100:0 (T100), 50 : 50 (T50), and 0 : 100 (T0). Samples were stained using 2 μL Hoescht 33342 (40 μLmL-1 in Dulbecco’s phosphate buffered saline), 3 μL of PI (0.5 mg mL-1 in PBS), 3 μL of CF (0.46 mg mL-1 in DMSO), and were incubated for 8 min at room temperature. After staining, the samples were placed on a slide, coverslipped, and evaluated immediately by epifluorescent microscopy. The Hoescht, PI, and CF fluorescence was detected using a filter with excitation at 352, 538, and 495 nm and emission at 455, 617, and 517 nm, respectively. Approximately 200 sperm cells per slide were examined and classified based on the fluorescence emitted from each probe. Spermatozoa CF+/IP- were considered as intact membranes, CF+/PI+ as acrosome membrane intact and plasma damaged, CF-/PI+ as damaged membranes, and CF-/PI- as acrosome membrane damaged and plasma intact. Hoeschst was used as positive dye. This experiment was replicated 6 times per group, and for statistical analyses, the data of plasma and acrosomal membrane integrity (dependent variables) in the treatments T0, T50, and T100 (independent variables) were submitted to simple linear regression analysis by STATVIEW software (SAS Institute Inc., Cary, NC, USA). The CFDA/PI probes were suitable for the analysis of acrosomal and membrane status of murine sperm and showed a high determination coefficient to plasma membrane integrity (R2 = 0.81; Y = 0.5412x + 6.375) and acrosome integrity (R2 = 0.85; Y = 0.5653x + 11.653). The described protocol was efficient for the simultaneous evaluation of plasma and acrosomal membrane integrity of murine spermatozoa, proving that CFDA can be employed to access acrosomal integrity as an alternative to FITC-PSA. Financial support: FAPESP.

Phosphate efflux as a test of plasma membrane leakage in Saccharomyces cerevisiae cells

2020 ◽  
pp. 1-5
Author(s):  
Ludmila Trilisenko ◽  
Airat Valiakhmetov ◽  
Tatiana Kulakovskaya
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Propidium Iodide ◽  
Membrane Integrity ◽  
Yeast Cells ◽  
Membrane Disruption ◽  
Uptake System ◽  
Plasma Membrane Integrity ◽  
Medium Time ◽  
The Difference

Plasma membrane integrity is a key to cell viability. Currently, the main approach to assessing plasma membrane integrity is the detection of penetration of special dyes, such as trypan blue and propidium iodide, into the cells. However, this method needs expensive equipment: a fluorescent microscope or a flow cytometer. Besides, staining with propidium iodide occasionally gives false-positive results. Here, we suggest the phosphate (Pi) leakage assay as an approach to assess the increase in permeability of the plasma membrane of yeast cells. We studied the dependence of phosphate efflux and uptake into Saccharomyces cerevisiae cells on the composition of the incubation medium, time, and ambient pH. The difference in optimal conditions for these processes suggests that Pi efflux is not conducted by the Pi uptake system. The Pi efflux in water correlated with the proportion of cells stained with propidium iodide. This indicated that Pi efflux is associated with cytoplasmic membrane disruption in a portion of the yeast cell population. The assay of Pi efflux was used to evaluate membrane disruption in S. cerevisiae cells treated with some heavy metal ions and detergents.

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