143. THE FIRST EVIDENCE OF HIGH SUSCEPTIBILITY TO COLD SHOCK BY THE SPERMATOZOA OF A MARSUPIAL, THE FAT TAILED DUNNART (SMINTHOPSIS CRASSICAUDATA)

2009 ◽  
Vol 21 (9) ◽  
pp. 61
Author(s):  
N. A. Czarny ◽  
J. C. Rodger
Keyword(s):  
Cold Shock ◽  
Egg Yolk ◽  
Significant Loss ◽  
Tasmanian Devil ◽  
Freezing And Thawing ◽  
Sminthopsis Crassicaudata ◽  
Captive Populations ◽  
Iced Water ◽  
Reproductive Techniques ◽  
Paradigm Shifting

Carnivorous marsupials are native Australian predators including the highly threatened northern quoll (Dasyurus hallucatus) and Tasmanian devil (Sarcophilus harrisii). These species are currently actively managed in captive populations but assisted reproductive techniques such as gamete banking may also contribute to their conservation. Previous studies on a model dasyurid, the fat tailed dunnart (Sminthopsis crassicaudata), have found that spermatozoa do not survive freezing and thawing using a variety of freezing protocols and cryoprotectants. We have re-examined cold shock to investigate problems with sperm cryopreservation in S. crassicaudata. Epididymal spermatozoa were rapidly cooled to 0.5ºC in a pre-cooled tube held in an iced water slurry and upon re-warming the spermatozoa were non-motile (n=6). The addition of up to 20% egg yolk, which is considered protective to the spermatozoa of cold shock sensitive eutherians, did not improve the outcome (n=6). Similarly when S. crassicaudata spermatozoa were rapidly cooled to 4ºC, just 2% remained motile upon re-warming (n=10). However when spermatozoa were combined with at least 10% egg yolk and rapidly cooled to 4ºC only small reductions in motility were observed upon rewarming (n≥8). In order to achieve motile spermatozoa at 0ºC, controlled rate cooling at 0.5ºC/minute was examined. In the absence of egg yolk there was a decline in the percentage of motile spermatozoa below 4ºC (n=6). However if spermatozoa were combined with at least 10% egg yolk there was no significant loss of motility at temperatures as low as 0ºC (n=6). This study has revealed that at least one species of marsupial is highly susceptible to cold shock. These paradigm shifting findings give direction to future experiments aiming to develop a robust technique for sperm preservation in endangered dasyurids.

The spermatozoa of the dasyurid marsupial, Sminthopsis crassicaudata, are highly susceptible to cold shock

2010 ◽  
Vol 22 (3) ◽  
pp. 580 ◽  
Author(s):  
N. A. Czarny ◽  
J. C. Rodger
Keyword(s):  
Experimental Study ◽  
Cold Shock ◽  
Egg Yolk ◽  
Significant Decline ◽  
Sperm Cryopreservation ◽  
Future Studies ◽  
Sminthopsis Crassicaudata ◽  
Iced Water ◽  
Epididymal Spermatozoa ◽  
The Impact

Since the late 1970s research has suggested that marsupial spermatozoa did not suffer cold shock. We have re-examined cold shock to investigate problems with freezing of spermatozoa from a dasyurid marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata). Epididymal spermatozoa were rapidly cooled to 0.5°C in a pre-cooled tube held in an iced-water slurry. Upon re-warming all spermatozoa were immotile and the addition of 10% or 20% egg yolk to the sperm medium had no beneficial effect. Spermatozoa that were rapidly cooled to 4°C maintained only 2% motility when re-warmed but the addition of at least 10% egg yolk was beneficial and upon re-warming greater than 65% of the initial motility was maintained. In order to achieve motile spermatozoa at 0°C, controlled-rate cooling at 0.5°C min–1 was examined. In the absence of egg yolk there was a significant decline in the percentage of motile spermatozoa below 4°C. However, the inclusion of at least 10% egg yolk resulted in no loss of motility in spermatozoa cooled to 0°C. This is the first experimental study indicating that spermatozoa from a marsupial are highly susceptible to cold shock and that the impact of rapid chilling can be mitigated by the addition of 10% egg yolk. The ability to successfully cool the spermatozoa of S. crassicaudata to 0°C may have an important role in future studies examining dasyurid sperm cryopreservation.

Freezing buck semen diluted in amine-organic buffers

1993 ◽  
Vol 56 (3) ◽  
pp. 387-391
Author(s):  
S. Dunner
Keyword(s):  
Cold Shock ◽  
Egg Yolk ◽  
Freezing And Thawing ◽  
Freezing Procedure

AbstractAmine-organic buffers (BESKOH and TESTRIS) were compared for their ability to be used for buck semen dilution in a freezing procedure. BESKOH showed better results (P < 0·05) at the time of dilution and after chilling at +5°C (72% motility v. 54% at dilution and 62% v. 39% after chilling). After freezing and thawing, none of the variables measured (motility, normal acrosome ridge and normal swelling) was significantly different. A washing procedure was necessary when freezing was undertaken and egg yolk addition was necessary to avoid cold shock as the temperature lowered during chilling. There were no significant differences between a 3% and 12% level of egg yolk addition.

220 LIVE BIRTH OF A MOHOR GAZELLE (GAZELLA DAMA MHORR) CALF FOLLOWING INTRAUTERINE INSEMINATION OF MOTHER WITH FROZEN - THAWED SEMEN

2006 ◽  
Vol 18 (2) ◽  
pp. 218 ◽  
Author(s):  
J. J. Garde ◽  
M. Gomendio ◽  
G. Espeso ◽  
E. R. S. Roldan
Keyword(s):  
Endangered Species ◽  
Artificial Insemination ◽  
Intrauterine Insemination ◽  
Live Weight ◽  
Egg Yolk ◽  
Freezing And Thawing ◽  
Technical Problems ◽  
In The Wild ◽  
Reproductive Techniques

Gazella dama mhorr is an endangered species, and no animals have been observed in the wild since 1968. Assisted reproductive techniques have been used to propagate endangered species. However, no live offspring has been produced after cryopreservation of semen from gazelle species. Therefore, the objective of this study was to evaluate whether cryopreserved Mohor gazelle spermatozoa can fertilize in vivo after artificial insemination. Semen was collected by electroejaculation from four males and centrifuged at 700g for 5 min at room temperature. The supernatant was discarded, and the semen pellet was resuspended with a TEST-1% egg yolk diluent containing 6% glycerol to provide 400 � 106 spermatozoa/mL. The extended semen was loaded into 0.25-mL plastic straws, cooled slowly to 5�C, and equilibrated at 5�C for 2 h. Straws were frozen in nitrogen vapors for 10 min and then plunged into liquid nitrogen. After thawing (37�C, 30 s), the effects of cryopreservation on sperm motility and acrosomal integrity were examined. Percentage of motile sperm in fresh samples was 88.7 � 3.8% (mean � SEM), decreased (P < 0.0001) to 58.7 � 3.8% after freezing and thawing, and then to 40.0 � 3.8% after 120 min incubation at 37�C. Spermatozoa with normal acrosomes decreased (P < 0.0001) after freezing and thawing (from 94.5 � 4.2% to 56.0 � 4.2%), but did not significantly decrease after sperm incubation. Frozen spermatozoa from two males were used in an intrauterine insemination trial. Estrus of females (n = 15) was synchronized with controlled internal drug release (CIDR, InterAg, Hamilton, New Zealand). Single CIDRs (type G, 330 mg progesterone/device) were inserted intravaginally for a total of 13 days. On Day 10, devices were replaced in each animal and all females received an injection of prostaglandin F2� (PGF2�; 125 mg/female). At CIDR withdrawal, females received 250-300 IU equine chorionic gonadotropin (eCG: Folligon; Intervet, Salamanca, Spain). After anesthesia with an intravenous injection of xylazine hydrochloride (Rompun; Bayer, Madrid, Spain; 0.8 mg/kg live weight) and ketamine hydrochloride (Imalgene; Leti & Merieux, Madrid, Spain; 2.0 mg/kg live weight), eight females were inseminated with 100 � 106 frozen-thawed spermatozoa 56-57 h after removal of the CIDRs, and seven were inseminated after 64-65 h. Females were inseminated directly into the uterus using laparoscopy. Anesthesia was reversed with yohimbine hydrochloride (0.3 mg/kg live weight). One female in the second group became pregnant. After a 202-day gestation, the female gave birth to one healthy Mohor gazelle male calf. These results demonstrate for the first time the successful use of frozen-thawed semen by means of artificial insemination for the rescue of endangered gazelle species. However, our results reveal that a number of unresolved technical problems remain to be addressed. This work was supported by the Spanish Ministry of Education and Science (REN2003-1587).

scholarly journals Observations on the viability of Trichomonas foetus during the process of freezing to –79° C. and thawing in the presence of glycerol

1956 ◽  
Vol 54 (3) ◽  
pp. 335-341 ◽  
Author(s):  
L. P. Joyner ◽  
G. H. Bennett
Keyword(s):  
Egg Yolk ◽  
Toxic Effects ◽  
Freezing And Thawing ◽  
Sperm Survival

1. It has been confirmed that Trichomonas foetus fails to survive freezing and thawing in the presence of 10 % glycerol in egg-yolk-citrate diluent. Under the same conditions adequate sperm survival was demonstrated.2. Trichomonads were particularly sensitive to the toxic effects of glycerol when suspended in egg-yolk citrate.3. Trichomonads will survive freezing and thawing when suspended in other diluents such as egg-yolk phosphate or milk.

Stability of Antibiotic Admixtures Frozen in Minibags

1977 ◽  
Vol 11 (9) ◽  
pp. 542-548 ◽  
Author(s):  
B. A. Dinel ◽  
D. L. Ayotte ◽  
R. J. Behme ◽  
B. L. Black ◽  
J. L. Whitby
Keyword(s):  
Significant Loss ◽  
Penicillin G ◽  
Freezing And Thawing ◽  
Ampicillin Sodium ◽  
Gentamicin Sulfate ◽  
Cefazolin Sodium ◽  
The Stability ◽  
Diffusion Assay ◽  
Percent Normal ◽  
Gel Diffusion

This study investigated the stability of frozen antibiotic admixtures prepared in minibags containing 50 ml of 0.9 percent normal saline and 5 percent dextrose in water. The minibag antibiotic admixtures studied were ampicillin sodium 1 g, carbenicillin disodium 2 g, cephalothin sodium 1 g, cloxacillin sodium 1 g, cefazolin sodium 1 g, gentamicin sulfate 50 mg, penicillin G potassium 2 million units, erythromycin gluceptate 500 mg and rolitetracycline base 275 mg. The stability of each antibiotic was determined by a quantitative microbiological agar gel diffusion assay. The minibags were frozen within an hour of the admixtures being prepared. During storage in a freezer at −20 °C (–4 °F), the minibags were placed in a storage module to reduce unnecessary handling. Assays were conducted on minibags which had been frozen for 24 hours, 7 days and 30 days. All thawed samples were re-assayed after storage at 5–6 °C (21–23 °F) for 21 hours. The results of the study indicated that minibag admixtures of carbenicillin disodium, cephalothin sodium, cloxacillin sodium, gentamicin sulfate, penicillin G potassium, erythromycin gluceptate, cefazolin sodium and rolitetracycline base in the concentrations tested can be frozen for 30 days without a significant loss in activity. These antibiotics were also stable when thawed and stored at 5–6 °C (21–23 °F) for 21 hours. Ampicillin sodium admixtures were not stable when frozen. A handling procedure for the frozen minibags is described. Specific recommendations are also included for freezing and thawing the minibags and in the use of a specially designed storage module.

scholarly journals Water Loss during Contracture of Muscle

1962 ◽  
Vol 46 (1) ◽  
pp. 131-142 ◽  
Author(s):  
Benjamin Kaminer
Keyword(s):  
Smooth Muscle ◽  
Water Loss ◽  
Significant Loss ◽  
Frog Muscle ◽  
Tetanic Stimulation ◽  
Force Of Contraction ◽  
Freezing And Thawing ◽  
Microscopic Studies ◽  
Relationship Of ◽  
The Relationship

The relationship of contracture and exudation of water in frozenthawed frog muscle was studied. With maximum shortening, there was a water loss of 35 per cent of the weight of muscle. By restricting the contraction, it was demonstrated that the amount of water loss was proportional to the degree of shortening, there being no significant loss with isometric contraction. Muscle already shortened by tetanic stimulation also exuded water on subsequent freezing and thawing. The force of contraction could be reduced by depleting the muscle of calcium and it was shown that the amount of water exuded was also proportional to the tensile ability of the muscle. In a smooth muscle (anterior byssus retractor of Mytilus) which did not contract vigorously only a little water exuded. Contracture produced by caffeine was similarly associated with a loss of water. Microscopic studies revealed a disruption of the sarcomeres of the frozen-thawed muscle which contracted; glycerol-extracted and calcium-depleted muscles, which did not contract on freeze-thawing, did not show such disruption. Freezing and thawing of actomyosin caused a reversible syneresis of the protein. It is concluded that the exudation of the water is not merely due to the freezing and thawing but is also dependent on the contractile events.

97 IMPROVED CRYOPRESERVATION OF DOMESTIC CAT SPERMATOZOA IN A SOY LECITHIN-BASED EXTENDER

2011 ◽  
Vol 23 (1) ◽  
pp. 153 ◽  
Author(s):  
M. M. Vick ◽  
H. L. Bateman ◽  
W. F. Swanson
Keyword(s):  
Sperm Motility ◽  
Room Temperature ◽  
Egg Yolk ◽  
Tris Buffer ◽  
Domestic Cat ◽  
Dna Integrity ◽  
Freezing And Thawing ◽  
Soy Lecithin ◽  
Acridine Orange Staining ◽  
Acrosome Integrity

Development of a chemically defined, plant-based cryopreservation media would reduce extender variability and the potential for transmission of zoonotic pathogens compared with traditional egg-yolk-based extenders. The objective of this study was to compare effects of egg yolk- and soy lecithin-based cryopreservation media and the temperature of glycerol addition on sperm parameters following freezing and thawing of domestic cat spermatozoa. Fresh semen was collected by manual stimulation on 3 separate occasions from 4 adult male cats. Each ejaculate was washed to remove seminal plasma, divided into 4 equal aliquots, and extended at room temperature in one of the following treatments: 1) TEST-egg yolk (Irvine Scientific Inc., Santa Ana, CA, USA) medium with 4% glycerol (EYG); 2) TEST-egg yolk, with 4% glycerol added after cooling to 5°C (EY); 3) TES-Tris buffer with soy-lecithin (1%) and 4% glycerol (SLG); and 4) TES-Tris buffer with 1% soy-lecithin, and 4% glycerol added after cooling to 5°C (SL). Sperm progressive motility (%) and rate of progressive movement (scale of 0–5) were evaluated at 0, 1, 3, 6, and 24 h post-thaw. Sperm capacitation (chlortetracycline staining), acrosome integrity (FITC-PNA staining), and DNA integrity (acridine orange staining) were assessed at 15 min post-thaw. Data were exponentially transformed to achieve normal distribution and then subjected to GLM analysis to determine effects of media and temperature of glycerol addition on sperm traits. At 0 and 1 h post-thaw, acrosome integrity, DNA integrity and % sperm motility did not differ (P > 0.05) among treatments. However, % sperm motility was greater in the soy-based media compared to egg yolk-based media at 3, 6, and 24 h post-thaw (Table 1; P < 0.05). A higher percentage of uncapacitated spermatozoa were present in soy-based compared to egg-yolk based cryopreservation media (63.9 ± 9.3 v. 51.2 ± 11.5, respectively; P < 0.05), regardless of temperature of glycerol addition. Finally, addition of glycerol at 5°C resulted in higher % sperm motility compared to room temperature at 6 and 24 h post-thaw in both medium types (Table 1; P < 0.05). Our results suggest that use of a chemically defined, soy-based medium improves long-term motility and capacitation status of frozen–thawed domestic cat spermatozoa compared with cryopreservation in a traditional egg yolk-based extender. Table 1.Motile spermatazoa and motility score at 3, 6, and 24 h

75 GLUCOSE CONCENTRATION OF FREEZING EXTENDER MODULATES THE TYROSINE PHOSPHORYLATION PATTERN OF FROZEN-THAWED BOAR SPERMATOZOA

2009 ◽  
Vol 21 (1) ◽  
pp. 138
Author(s):  
J. E. Rodríguez-Gil ◽  
M. Hernández ◽  
M. M. Rivera ◽  
L. Ramió-Lluch ◽  
J. Ballester ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Tyrosine Phosphorylation ◽  
Glucose Concentration ◽  
Egg Yolk ◽  
Freezing And Thawing ◽  
Precise Control ◽  
Boar Sperm ◽  
Phosphorylation Pattern ◽  
Thawing Process ◽  
Boar Spermatozoa

The optimization of freezing extenders is an essential issue for enhancing boar sperm cryosurvival. The aim of the present study was to disclose the role of glucose concentration of freezing extender on the metabolic activity of frozen–thawed spermatozoa. To achieve it, pooled sperm-rich ejaculate fractions from 5 mature and fertile boars (3 ejaculates per boar) were collected using the gloved-hand method. After centrifugation (2400g for 3 min), the sperm pellet was split into 7 aliquots. The aliquots were diluted to a final concentration of 1 × 109 sperm mL–1, in a Tris-citric extender supplemented with 20% egg-yolk, 3% glycerol, and 0, 0.05, 2, 4, 10, 55, or 185 mm glucose. All the extenders were adjusted to a pH of 6.8 and 310 mOsm kg–1 to avoid osmolarity effects. Extended semen samples were dispensed into 0.5-mL straws, and frozen in a programmable cell freezer at 20°C min–1. Thawing was carried out in a water bath at 37°C for 20 s. Afterward, an analysis of protein phosphorylation in tyrosine residues was carried out through bi-dimensional electrophoresis followed by a Western blot analysis. This analysis indicated that sperm samples frozen in extenders without glucose showed specific changes in the tyrosine phosphorylation pattern compared with fresh sperm. Furthermore, the addition of glucose in increasing concentrations to the freezing extender was accompanied by a concentration-dependent decrease in the overall tyrosine phosphorylation pattern, especially in proteins with a molecular weight ranging from 150 to 200 kDa and an acidic isoelectric point (pI). The maximal decrease was observed in spermatozoa frozen in the extender containing 185 mm glucose, in which an additional decrease in the tyrosine phosphorylation of proteins ranging from 60 to 80 kDa, and a basic pI was also observed. These results suggest that glucose is a modulator in the resistance of boar sperm to support freezing and thawing process, because the precise protein phosphorylation pattern of spermatozoa is directly linked to their functional status. In this way, a precise control of the glucose concentration of the freezing extender would be required to improve boar sperm cryoresistance. Supported by CICYT (AGL2005-00760 and AGL2004-04756-C02-02/GAN), Madrid and GERM (04543/07), Murcia, Spain.

Sign in / Sign up

Export Citation Format

Share Document