scholarly journals Generation of live rat offspring by intrauterine insemination with epididymal spermatozoa cryopreserved at -196 degrees C

Reproduction ◽  
2001 ◽  
pp. 463-467 ◽  
Author(s):  
E Nakatsukasa ◽  
T Inomata ◽  
T Ikeda ◽  
M Shino ◽  
N Kashiwazaki
Keyword(s):  
Intrauterine Insemination ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Physical Stress ◽  
Female Rats ◽  
Freezing And Thawing ◽  
Cryoprotective Agents ◽  
Epididymal Spermatozoa ◽  
Normal Offspring ◽  
Successful Production

This study reports the development of a reliable method for cryopreservation of rat epididymal spermatozoa and the production of live young by artificial insemination using these cryopreserved spermatozoa. The motility and membrane integrity of rat spermatozoa were investigated after spermatozoa had been subjected to physical stress and frozen with various concentrations of glycerol (0, 3 and 6%) either in the presence or absence of Equex Stem as cryoprotective agents. The ability of cryopreserved spermatozoa to generate normal offspring by intrauterine insemination was also evaluated. Rat spermatozoa that had been centrifuged at 700 g for 5 min showed a significant decrease in motility compared with non-centrifuged spermatozoa. In addition, after centrifugation three times the percentage of membrane-intact spermatozoa decreased to approximately 0%. The percentage of membrane-intact spermatozoa was significantly higher (P < 0.01) in semen samples that had been frozen in medium without glycerol than in samples frozen in medium with 3% glycerol. Although the addition of 0.7% Equex Stem to medium without glycerol or with 3% glycerol did not influence rates of sperm motility after freezing and thawing, the percentage of membrane-intact spermatozoa was improved by the presence of 0.7% Equex (P < 0.05). Therefore, rat spermatozoa were handled gently to avoid physical stress and were frozen in medium containing 23% egg yolk, 8% lactose monohydrate and 0.7% Equex Stem, at pH 7.4 adjusted with 10% Tris(hydroxymethyl)aminomethane solution. Thirteen female rats were inseminated into the oviductal end of both uterine horns with frozen-thawed spermatozoa. Forty-one normal live offspring were obtained from nine of the inseminated females. These results indicate that frozen-thawed rat spermatozoa can generate normal offspring. To our knowledge, this procedure is the first successful production of offspring using spermatozoa cryopreserved in liquid nitrogen.

12 GENERATION OF RAT OFFSPRING DERIVED FROM CRYOPRESERVED SPERMATOZOA IN JAPANESE NATIONAL BIORESOURCES

2007 ◽  
Vol 19 (1) ◽  
pp. 124 ◽  
Author(s):  
N. Kashiwazaki ◽  
Y. Seita ◽  
K. Naoi ◽  
A. Takizawa ◽  
T. Kuramoto ◽  
...  
Keyword(s):  
Genetic Resources ◽  
Intrauterine Insemination ◽  
Egg Yolk ◽  
Laboratory Animals ◽  
Freezing And Thawing ◽  
Rat Strains ◽  
High Pregnancy Rate ◽  
Cryopreserved Sperm ◽  
Humidified Air ◽  
Thawing Procedure

The purpose of the National BioResource project is to facilitate the availability of genetically and phenotypically standardized rat strains for life sciences. The BioResource is available to scientists worldwide. To bank genetic resources efficiently in the rat, cryopreservation of both sperm and embryos is a very important technology. The objective of the present study was to confirm the ability of banked and transported rat spermatozoa to fertilize oocytes through intrauterine insemination and for the embryos to develop to term, with the ultimate aim of developing a system for banking rat genetic resources. The epidydimal spermatozoa from the KLM rat, whose body size is small because the Prkg2 gene is partially defective, were frozen with egg yolk medium supplemented with 0.7% Equex Stm (Nakatsukasa et al. 2001 Reproduction 122, 463–467) and banked in the Institute of Laboratory Animals, Kyoto University. The cryopreserved sperm in 0.25-mL straws were transported to the laboratory at Azabu University, Kanagawa. Two straws from different males were thawed in a 37�C water bath for 15 s. Thawed semen was diluted with 1.0 mL of mR1ECM (Miyoshi et al. 1997 Biol. Reprod. 56, 180–185) with 0.4% (w/v) bovine serum albumin (BSA, fraction V; Sigma-Aldrich Japan K.K., Tokyo, Japan) at 37�C and then incubated at 37�C in 5% CO2 in humidified air until insemination. The percentage of motile spermatozoa was assessed visibly and determined by direct observation at 37�C under a light microscope at 100�. The thawed semen (50 �L, 3–4 � 105 sperm cells) was then inseminated into the top of both uterine horns of recipient females that were mated with a vasectomized male. The post-thaw motility of frozen spermatozoa was 10%. Seven of 15 inseminated females became pregnant and 13 live pups were born. It is thought that the low number of pups born in spite of the relatively high pregnancy rate was caused by sperm damage during the freezing and thawing procedure. The results of the present study show that rat spermatozoa cryopreserved in the BioResource have the ability to revive genetic resources through intrauterine insemination.

113 EFFECT OF SPERM COATING ON THE QUALITY OF BOVINE FROZEN - THAWED SPERMATOZOA

2006 ◽  
Vol 18 (2) ◽  
pp. 164
Author(s):  
M. Thys ◽  
A. Van Soom ◽  
J. Dewulf ◽  
T. Rijsselaere ◽  
A. de Kruif
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Univariate Analysis ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Freezing And Thawing ◽  
Bovine Sperm ◽  
Sperm Characteristics ◽  
Group 2 ◽  
Group 1

The substantial decrease of sperm quality after cryopreservation remains an important issue in the artificial insemination industry. Sperm coating with Triladyl® (Minitübe, Tiefenbach, Germany) during ejaculation can preserve sperm characteristics and oocyte penetrating capacity of fresh bovine spermatozoa stored in egg yolk diluent for up to 6 days (De Pauw et al. 2003 Theriogenology 59, 1109–1122). Since collecting semen in a tube containing egg yolk-Tris extender (sperm coating) limits the period of contact between spermatozoa and seminal plasma, the present experiment was conducted to assess if this slightly adjusted method of sperm collection could also have a significant effect on bovine sperm quality after cryopreservation. Semen of five young Holstein Friesian bulls was collected by means of an artificial vagina connected to an empty tube (Group 1; five ejaculates per bull) or a tube containing 4 mL of an egg yolk-Tris extender (Groups 2 and 3; each five ejaculates per bull). The semen samples of Group 1 were conventionally diluted in straws (60 × 106 sperm/mL), frozen, and stored in liquid nitrogen. The samples of Group 3 were centrifuged, and after removing diluent and seminal plasma, the sperm pellet was conventionally diluted and processed. The samples of Group 2 were processed without removal of the supernatant. After thawing each ejaculate was analyzed for average path velocity (VAP), beat cross frequency (BCF), and progressive motility (PROG) using CASA (Minitübe, Tiefenbach, Germany). Furthermore, the membrane integrity of each sample was evaluated using fluorescent SYBR®–14/PI staining (BD Biosciences, Erembodegem, Belgium). All parameters were compared among the three groups of sperm using univariate analysis of variance (SPSS 12.0; SPSS, Inc., Chicago, IL, USA). No significant differences could be observed among the three groups for all of the evaluated sperm characteristics (Table 1). A significant effect of the bull could be determined for all analyzed parameters (P ≤ 0.02), except for the percentage of moribund cells. Nevertheless, the group-bull interaction was never statistically significant. Coating bovine sperm with an egg yolk-Tris extender during ejaculation cannot prevent the substantial deterioration of the spermatozoa that occurs during freezing and thawing since this method of sperm collection does not significantly influence the motility parameters or the membrane integrity after thawing. Table 1. VAP, BCF, PROG, and percentage of membrane-intact, dead, and moribund spermatozoa for the three groups of sperm This research was supported by IWT (no. IWT/020727).

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).

76 SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION IN EXTENDERS WITH OR WITHOUT EGG YOLK

2009 ◽  
Vol 21 (1) ◽  
pp. 138
Author(s):  
J. R. Saenz ◽  
C. Dumas ◽  
B. L. Dresser ◽  
M. C. Gómez ◽  
R. A. Godke ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Egg Yolk ◽  
Human Sperm ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
Santa Ana ◽  
Sperm Suspension ◽  
Epididymal Spermatozoa ◽  
One Way Anova

Our purpose was to compare in vitro survivability and functionality of cat epididymal spermatozoa cryopreserved in TEST egg-yolk buffered extender (TYB) with that obtained by use of clear Tris-citrate and HEPES-buffered extenders containing BSA. Testes were transported to the lab in HEPES saline; epididymides were dissected in HEPES-199 medium (HE-199) and repeatedly sliced. The sperm suspension was filtered (40 μm), layered onto a density gradient column (Isolater, Irving Scientific, Santa Ana, CA), and centrifuged at 600g for 20 min. Aliquots of the sperm pellet were extended in TYB, Human Sperm Preservation Medium (HSPM), or Tris-citrate + 10% BSA (TCBSA). After cooling to 4°C, samples were diluted 1:1 with extender + 12% glycerol in 4 steps as modified from Gao DY et al. 1995 Hum. Reprod. 10, 1109–1122. Then, samples were loaded into 0.25-mL straws, sealed, and frozen on a dry ice block (–80°C) for 20 min before storage in LN2. Straws were thawed by exposure to air (22°C) for 5 s and immersion in a 60°C water bath for 5 s. Samples were diluted by addition of HE-199 in 7 steps as modified from Gao DY et al. 1995 Hum. Reprod. 10, 1109–1122, centrifuged at 200g for 10 min and pellets resuspended in HE-199. Motility (MOT, phase contrast, 37°C), membrane integrity (MI, SYBR 14–PI), and acrosomal status (AS, FITC–PNA) were evaluated at 0 h, after gradual cooling to 4°C, and after freezing at 0 h and 3 h post-thaw (37°C). Cumulus oocyte complexes (COC) were placed in modified TCM-199 and cultured for 24 h in 5% O2, 5% CO2, and 90% N2 at 38°C (IVM). For IVF, COC were co-incubated with spermatozoa frozen in either TYB or HSPM in droplets (1 million sperm mL–1) of IVF medium under 5% CO2 in air at 38°C. After 18 h, oocytes were rinsed and cultured using a 3-step system (Pope CE et al. 2006 Theriogenology 66, 59–71) until blastocyst development was evaluated (Day 8). There were no treatment differences at any time/temperature point for the 3 sperm parameters evaluated (one-way ANOVA; P > 0.05). As shown in Table 1, sperm motility in TCBSA and HSPM decreased by 20% after cooling to 4°C and another 20% after freezing, whereas motility in TYB was maintained after cooling and decreased <30% after freezing. Membrane integrity and acrosomal status values were 12 to 15% greater at collection, at 4°C and at 0 h post-thaw, and 25% greater at 3 h post-thaw than were the motility values. Cleavage frequency and blastocyst development rate of 203 IVM oocytes after IVF using sperm frozen in TYB and HSPM was 36 v. 33% and 50 v. 44%, respectively. In summary, we have shown that cat epididymal spermatozoa can be frozen successfully in cryoprotectant solutions that do not contain egg yolk. Table 1.Motility, membrane integrity and acrosomal status of cat epididymal sperm after cryo-storage

112 EFFECT OF EGG YOLK CONCENTRATION IN TEST BUFFERED EXTENDER ON SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION

2010 ◽  
Vol 22 (1) ◽  
pp. 214
Author(s):  
J. R. Saenz ◽  
C. Dumas ◽  
B. L. Dresser ◽  
M. C. Gómez ◽  
R. A. Godke ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Egg Yolk ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
Santa Ana ◽  
Sperm Suspension ◽  
Epididymal Spermatozoa ◽  
One Way Anova ◽  
Cryoprotectant Solution

Our purpose was to examine the effect of egg yolk concentration (EY; 2, 5, or 10%) on in vitro survivability and functionality of cat epididymal spermatozoa cryopreserved in TEST-buffered extender (TYB). Testes were transported in HEPES saline; epididymes were dissected in HEPES 199 medium (He199) and repeatedly sliced. The sperm suspension was filtered (40 μ), layered onto a density gradient column (Isolate®, Irving Scientific, Santa Ana, CA, USA), and centrifuged at 650 g for 20 min. Aliquots of the sperm pellet were extended in TYB containing 2, 5, or 10% EY. After cooling to 4°C, samples were diluted 1:1 with TYB containing 2, 5, or 10% EY + 12% glycerol in 4 steps as modified from Gao DY et al. Hum. Reprod. 1995 10, 1109-1122. Then, samples were loaded into 0.25-mL straws, sealed, and frozen on a dry ice block (-80°C) for 20 min before storage in LN2. Straws were thawed by exposure to air (˜22°C) for 5 s and immersion in a 60°C water bath for 5 s. Samples were diluted by addition of He199 in 7 steps as modified from Gao DY et al. Hum. Reprod. 1995 10, 1109-1122 centrifuged at 200g for 10 min, and pellets resuspended in He199. Motility (Mot, phase contrast, 37°C), membrane integrity (M.I., SYBR 14-PI), and acrosomal status (A.S., FITC-PNA) were evaluated at 0 h, after gradual cooling to 4°C and after freezing at 0 and 3 h post-thaw (37°C). Ten replicates were done. Cumulus oocyte complexes (COC) were placed in modified TCM-199 and cultured for 24 h in 5% O2, 5% CO2, and 90% N2 at 38°C (IVM). For IVF, COC were co-incubated with spermatozoa frozen in TYB + 2% egg yolk or HSPM (no egg yolk) in droplets (1 million sperm/mL) of IVF medium under 5% CO2 in air at 38°C. After 18 h, oocytes were rinsed and cultured until blastocyst development was evaluated (Day 8). There were no treatment differences at any time or temperature point for the 3 sperm characteristics evaluated (one-way ANOVA; P > 0.05). As shown in the Table 1, at 0 h post-thawing, sperm in each group retained ˜70% of their initial pre-freeze motility. After 3 h of post-thaw incubation, motility decreased to ˜50% of the pre-freeze value. Cooling to 4°C did not affect membrane integrity or acrosomal status, but post-thaw values were reduced by 30-35% as compared with pre-freeze. Cleavage frequency and blastocyst development of 284 IVM oocytes after IVF using sperm frozen in TYB + 2% EY and HSPM were 53 v. 52% and 42 v. 38%, respectively (P > 0.05). In summary, we have shown that cat epididymal spermatozoa can be frozen successfully in a cryoprotectant solution containing minimal egg yolk (2%). Table 1.Motility, membrane integrity, and acrosomal status of cat epididymal sperm after cryo-storage

95 EFFECT OF CRYOPROTECTANT ADDITION AND REMOVAL AT 4°C ON MOTILITY AND PLASMA MEMBRANE INTEGRITY OF BOVINE CAUDAL EPDIDYMAL SPERMATOZOA

2006 ◽  
Vol 18 (2) ◽  
pp. 156
Author(s):  
C. Guerrero ◽  
S. Leibo ◽  
D. Paccamonti ◽  
B. Eilts ◽  
K. Bondioli ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Single Step ◽  
Freezing Process ◽  
Computer Assisted ◽  
Chemical Toxicity ◽  
Plasma Membrane Integrity ◽  
Epididymal Spermatozoa

Cryopreservation of spermatozoa harvested from the epididymides would be a means of salvaging germplasm from genetically valuable males that die unexpectedly from injury, disease, or poaching. It is well known that the addition of cryoprotective agents (CPAs) is essential for sperm survival following the freezing process. However, CPAs can cause loss in sperm viability due to osmotic damage or chemical toxicity. The objective of this study was to determine the effects of single-step addition and/or removal of glycerol (GLY) or ethylene glycol (EG) on motility and plasma membrane integrity of bovine epididymal spermatozoa. Paired testes were obtained from mature bulls (n = 10) at a local abattoir and transported to the laboratory at 25–28°C within 4–6 h post-mortem. Epididymal spermatozoa were harvested by multiple incisions from the caudae epididymides of each bull, pooled, and washed in Brackett-Oliphant medium by centrifugation for 5 min at 500g. Pellets were resuspended in egg yolk-Tris-glucose-citric acid monohydrate medium (EYT-GC) at a concentration of 120 × 106 cells/mL and cooled to 4°C at a rate of 0.1°C/min. Specimens were allocated to each of five treatment groups: control (no CPA), 7% GLY, and 14% GLY, 7% EG, 14% EG. Then, replicate samples were diluted 1:1 in EYT-GC medium containing twice the final desired concentration of CPA. After being exposed for 10 min, each sample was diluted directly into EYT-GC at 4°C. Motility was assessed by means of a computer assisted semen analysis system and plasma membrane integrity was determined by SYBR 14 and propidium iodide staining followed by fluorescence microscopy. Differences among treatments were analyzed using one way ANOVA (P < 0.05). The results (Table 1) show that maximum survival, as assessed by measurements of motility and membrane integrity, was achieved with spermatozoa exposed to 7% EG. Almost identical intermediate levels of survival were observed with spermatozoa exposed to 7% GLY or 14% EG. The lowest survival was observed for spermatozoa exposed to 14% GLY. The results indicate that the use of EG as a cryoprotectant may minimize toxicity and osmotic damage to fresh bovine epididymal spermatozoa. Its efficacy as a CPA is currently being determined. Table 1. Sperm motility and membrane integrity (mean ± SEM) after addition of CPA to epididymal sperm

scholarly journals Changes in motility, morphology, plasma membrane and acrosome integrity during stages of cryopreservation of buck sperm

2014 ◽  
Vol 85 (1) ◽  
Author(s):  
Mushtaq Ahmad ◽  
Rashad Nasrullah ◽  
Hasan Riaz ◽  
Abdul Sattar ◽  
Nasim Ahmad
Keyword(s):  
Plasma Membrane ◽  
Sperm Morphology ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Structure And Function ◽  
Freezing And Thawing ◽  
Plasma Membrane Integrity ◽  
Acrosome Integrity ◽  
Live Sperm ◽  
And Function

Changes in sperm structure and function occur during the processing of semen. The present study was designed to investigate the effect on buck sperm during different stages of semen preparation including dilution, cooling, equilibration and freeze-thawing. Semen ejaculates from three mature bucks (replicates = 5) were diluted with tris-citric acid egg yolk glycerol extender at 37 ºC, cooled to 4 ºC over 90 min, equilibrated at 4 ºC for 2 h, transferred to 0.5 mL straws, placed in nitrogen vapour, frozen and thawed and then analysed. Sperm samples were assessed for percentage motility, acrosomal and plasma membrane integrity, live sperm, and morphology after dilution, cooling, equilibration and thawing. Mean percentage motility after dilution (86.0 ± 1.4%) was reduced significantly (p < 0.05) due to cooling and equilibration (77.6 ± 1.3% and 74.6 ± 1.4% respectively); furthermore, it decreased significantly (p < 0.05) after freezing and thawing (42.3 ± 2.5%). Mean percentage of live sperm was higher (p < 0.05) after dilution (89.3 ± 1.4%)compared with cooling (84.8 ± 1.8%) and equilibration (80.2 ± 2.5%) and further reduced (p < 0.05) after freezing and thawing (56.0 ± 3.4%). Sperm morphology dropped significantly (p < 0.05) from 96.4 ± 0.3% after dilution to 88.8 ± 1.3% at cooling and further decreased (p < 0.05) after freezing and thawing (81 ± 1.9%). Mean percentage of sperm with normal plasma membrane after dilution (82.2 ± 1.1%) was significantly reduced (p < 0.05) at cooling or equilibration (73.8 ± 1.8) and further decreased (p < 0.05) after freezing and thawing (50.1 ± 2.9%). The percentage of sperm with normal acrosomes did not differ significantly due to dilution, cooling or equilibration (85.8 ± 1.7%, 83.2 ± 1.6%, 81.7 ± 1.8%) but was significantly reduced after freezing and thawing (45.2 ± 2.8%). In conclusion, frozen thawed sperm showed maximum damage to motility, morphology, plasma membrane and acrosome integrity following cooling.

129 IMPROVEMENT OF BOAR SPERM CRYOSURVIVAL BY THE OPTIMIZATION OF CRYOPRESERVATION CONDITIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 182
Author(s):  
J. Roca ◽  
M. Hernandez ◽  
T. Cremades ◽  
J. M. Vazquez ◽  
E. A. Martinez
Keyword(s):  
Propidium Iodide ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Limiting Factors ◽  
Commercial Application ◽  
Glycerol Concentration ◽  
Freezing And Thawing ◽  
Warming Rate ◽  
Boar Spermatozoa

One of the most important limiting factors for the efficient commercial application of frozen–thawed semen on pig artificial insemination programs is the significant and consistent inter-boar variability in sperm cryosurvival. The objective of the present study was to evaluate the effectiveness of different cryopreservation conditions (CCs) for freezing and thawing boar spermatozoa, and to determine their suitability for individual boars, with particular reference to those that showed an intrinsic poor sperm cryosurvival. Using a split-ejaculate technique, single ejaculates from 53 boars were suspended in lactose-egg yolk extender containing 2 or 3% final glycerol concentration, packaged in 0.5-mL straws, cooled at rates of 10, 40, or 60�C min-1 using a programmable cell freezer, and stored in liquid nitrogen; the frozen samples were warmed at ≈1200�C min-1 (37�C water bath for 20 s) or ≈1800�C min-1 (70�C for 8 s). The cryopreservation condition including 2% glycerol, 40�C min-1 of cooling, and ≈1200�C min-1 of warming was considered as the control. Frozen–thawed sperm were evaluated at 30 and 150 min post-thawing for sperm motility (CASA system), plasma membrane integrity (SYBR-14 and propidium iodide), and acrosome membrane integrity (FITC-peanut agglutinin and propidium iodide). Data were analyzed using 2 different ANOVA mixed models. Whereas cooling rate had no influence (P ≥ 0.05), glycerol concentration and warming rate, both independently, affected (P ≤ 0.05) all post-thawing sperm assessments. No interaction (P ≥ 0.05) among effects was detected for any of the sperm parameters assessed. Evaluating the combined effect of glycerol concentration and warming rate, the highest post-thaw sperm quality was achieved from the semen samples frozen with 3% of glycerol and thawed at ≈1800�C min-1. Significant differences (P ≤ 0.05) among ejaculates (boars) to support the different CCs were shown in all post-thaw sperm assessments. Three different (P ≤ 0.05) ejaculate (boar) populations, defined by PATN analysis (PATN software package, CSIRO, Canberra, Australia), were identified according to post-thaw sperm assessments in semen samples frozen and thawed using control CC (populations so-called 'good', 'moderate', and 'bad' sperm freezers). Different (P ≤ 0.05) susceptibility in the tolerance of spermatozoa to support the different CCs was found among the ejaculate populations. Whereas spermatozoa from ejaculates considered as 'good' freezers are relatively unaffected (P ≥ 0.05), those from 'moderate' and, mainly, 'bad' freezers are very sensitive (P ≤ 0.05) to the modifications in the CCs. In conclusion, slight modifications in the CCs — glycerol concentration and warming rate for thawing — can improve the sperm cryosurvival of some ejaculates (boars), the improvement being particularly larger in those ejaculates (boars) classified as ' bad' sperm freezers. This work was supported by CICYT (AGF2005-00760), Madrid, Spain.

208EFFECT OF REFRIGERATION AND CRYOPRESERVATION ON THE QUALITY OF LION EPIDIDYMAL SPERMATOZOA

2004 ◽  
Vol 16 (2) ◽  
pp. 225 ◽  
Author(s):  
A.F. Malo ◽  
F. Martinez-Pastor ◽  
F. Olivier ◽  
T. Spies ◽  
E.R.S. Roldan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Quality ◽  
Osmotic Shock ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Storage Period ◽  
Egg Yolk ◽  
Plasma Membrane Integrity ◽  
Sperm Plasma Membrane ◽  
Epididymal Spermatozoa

Epididymal spermatozoa from harvested wild animals is potentially useful for conservation purposes, as it can be used for subsequent artificial insemination or stored in Biological Resource Banks for future use. The potential of sperm banking is of particular interest for use in lion (Panthera leo) populations maintained in small National Parks, as translocation of males to effect gene-flow is often problematic, resulting in the translocated lion being killed by resident pride males. We measured the change in sperm quality over time during cool storage (at 4°C) and after thawing of samples cryopreserved at −196°C. Also, we present a correlation between sperm plasma membrane integrity and mitochondrial activity as measured by fluorescent analysis. The testes from a pride lion were removed and transported to the laboratory (at 4°C) within 6h. The epididymides were removed and both cauda epididymides were flushed with 1mL of Tris-citrate egg yolk extender (Fraction A, Biladyl, Minitub, Germany). The sample containing 2930×106 cells mL−1 was washed (20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH;; 400mOsm/kg, pH 7; Sigma, South Africa) and after centrifugation (5min. at 600g), the pellet was resuspended in 0.5mL of washing solution (with 197mM NaCl instead of sucrose). One aliquot of spermatozoa was kept at 4°C and evaluated at 24h intervals for 7 days. A second aliquot of the sperm sample was extended in Tris-citrate egg yolk extender with glycerol (Fraction B, Biladyl), frozen in liquid nitrogen (LN) vapor and stored in LN. The frozen sample was later thawed and evaluated as for the cooled samples. Percentages of motile (MS) and progressive (PS) spermatozoa were assessed using a phase contrast microscope (×200; stage at 37°C). Sperm plasma membrane damage was assessed by determining the percentage of cells exhibiting red fluoresence after staining with propidium iodide (PI, 50ng/mL; 10min RT). Spermatozoa that did not stain red in PI were classified as plasma membrane intact (PMI). Resilience to hypo-osmotic shock and plasma membrane integrity were evaluated by incubating a portion of the sample in a 100mOsm/kg solution (10nM glucose, 20nM HEPES, 30nM NaCl) containing PI for 15min at room temperature. The percentage of sperm cells with active mitochondria (MIT) was determined by counting spermatozoa showing orange fluoresence over the mid-piece after staining with JC-1(7.5 uM Sigma) for 30min at 37°C. At collection, MS was 15% and did not show a significant decrease during the 7-day storage period. Initially, PS was 10% and dropped to 5% after 7 days, with values fluctuating during the storage period. Both PMI and HOSPMI were 80% on Day 1, gradually decreasing to 75% on Day 7 of storage. PMI and MIT showed a highly significant correlation (r=0.88; P=0.003; n=8). In frozen-thawed sperm samples, MS fell from a pre-freeze value of 15% to 5% after thawing. Similarly, PS fell from 10% in pre-freeze to 3% in frozen-thawed samples. Likewise, PMI, HOSPMI and MIT values were 80% and 45%, 87% and 45% and 89% and 49%, respectively. Our study showed that lion sperm PMI and MIT remained high after 7 days at 4°C. MS and PS, although low, did not vary during this same period. PI and JC-1 showed a significant correlation, suggesting that both might be affected by the same deleterious factors. Although PMI, HOSPMI and MIT values decreased approximately 40% after freezing, we feel that such sperm samples could be used for in vitro embryo production, if not by IVF, by ICSI. Of course, additional studies are needed to validate our suggestion.

Kriopreservasi Semen Kambing Boer dengan Konsentrasi Pengencer Nira Aren dan Gliserol Berbeda

2019 ◽  
Vol 6 (1) ◽  
pp. 1
Author(s):  
Muhammad Riyadhi ◽  
Anis Wahdi ◽  
Muhammad Rizal
Keyword(s):  
Sperm Motility ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Boer Goat ◽  
Palm Juice ◽  
Sperm Membrane ◽  
Live Sperm

ABSTRAK                                                                        Penelitian bertujuan untuk mengetahui efektivitas nira aren sebagai pengencer alternatif dalam proses pembekuan (kriopreservasi) semen kambing boer.Kriopreservasi semen kambing boer menggunakan pengencer tris-gliserol-kuning telur (P1 73-7-20%), nira aren-gliseol-kuning telur(masing-masing P2 74-6-20%, P3 73-7-20%, dan P4 72-8-20%) dan andromed (P5 tanpa mengandung kuning telur dan gliserol). Parameter evaluasi meliputi motilitas, viabilitas, dan membrane plasma utuh setelah pengenceran, ekuilibrasi dan thawing.  Evaluasi motilitas pasca thawing menunjukkan P5 52% berbeda nyata (P<0.05) dengan P1 42%, selanjutnya P5 dan P1 berbeda sangat nyata (P<0.05) dengan P2 8%, P3 6% dan P4 12%.  Viabilitas pasca thawing menunjukkan P5 65,4% tidak berbeda nyata (P>0,05) dengan P1 61,8%, akan tetapi P5 dan P1 berbeda sangat nyata (P<0.05) dengan P2 26,2%, P3 29,8%, dan P4 34%.  Membran plasma utuh (MPU) pasca thawing menunjukkan P5 66,2% tidak berbeda nyata (P>0,05) dengan P1 65,4%, akan tetapi keduanya berbeda sangat nyata (P<0.05) dengan P2 39%, P3 38%, dan P4 36,2%.  Disimpulkan kriopreservasi semen kambing boer dengan pengencer nira aren dan gliserol pada konsentrasi berbeda belum dapat dipergunakan sebagai sumber bibit berdasarkan standar nasional Indonesia.Kata Kunci : Kambing boer, semen, nira arenABSTRACTThe experiment was conducted to determine the effect of sugar palm juice as alternative extender for cryopreservation process of boer semen.Tris-glycerol-egg yolk (P1 73-7-20%), Sugar palm juice-glyserol-egg yolk (P2 74-6-20%, P373-7-20%, dan P4 72-8-20%), and andromed (P5) used as a extender  in the cryopreservation process of boer semen.  Sperm motility (%), live sperm (%) and sperm membrane integrity (%) were recorded after diluted, equilibration and freeze-thawing.  Result of post thawing motility showed that P5 52% was significantly different (P <0.05) with P1 42%, then P5 and P1 were significantly different (P <0.05) with P2 8%, P3 6% and P4 12%. Viability after thawing showed P5 65.4% was not significantly different (P> 0.05) with P1 61.8%, but P5 and P1 significantly different (P <0.05) with P2 26.2%, P3 29.8 %, and P4 34%. Spermmembrane integrity post-thawing showed P5 66.2% was not significantly different (P> 0.05) with P1 65.4%, but both were very significantly different (P <0.05) with P2 39%, P3 38% and P4 36.2%. Conclusions, sugar palm juice-glycerol-egg yolk with differentconcentrationsineffectively as an alternative extenderin cryopreservation of boer semen.Keywords: boer goat, semen, sugar palm juice

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