215 EPIDIDYMAL SPERM CRYOPRESERVATION OF ONE SOMALIA WILD ASS (EQUUS AFRICANUS SOMALIENSIS) USING SIX DIFFERENT EXTENDERS

2006 ◽  
Vol 18 (2) ◽  
pp. 215 ◽  
Author(s):  
M. Álvarez ◽  
F. Martínez-Pastor ◽  
V. García-Macías ◽  
S. Borragán ◽  
M. Celada ◽  
...  
Keyword(s):  
Species Differences ◽  
Sperm Concentration ◽  
Egg Yolk ◽  
Red List ◽  
Biological Resource ◽  
Epididymal Sperm ◽  
Sperm Cryopreservation ◽  
The Poor ◽  
Wild Ass ◽  
Viable Sperm

The Somalia wild ass (Equus africanus somaliensis) is a critically endangered taxon (IUCN 2004 red list) which could benefit from biological resource banking. In this work, we studied the effect of different extenders applied to the cryopreservation of epididymal sperm obtained from one male of this subspecies. This animal (13 years old; housed in Cabarceno Park, Cantabria, Spain) was castrated because of very aggressive behavior with other mature males. Genitalia were dissected and weighed (testicles: right, 166 g, and left, 179 g; cauda epididymis: right, 9.3 g, and left, 11.8 g). Sperm were flushed from the cauda epididymis, yielding 15 mL of sample. Sperm concentration was 15 × 109 spermatozoa/mL, totaling 225 × 109 (allowing 4500 doses at 50 × 106 sperm/dose). Sperm motility (TM = % total motile; PM = % progressive; VAP = average path velocity) was assessed by CASA (Microptic, Barcelona, Spain). Viability (VIAB = % viable sperm) and acrosomal status (ACR = % viable spermatozoa with intact acrosomes) were assessed using propidium iodide (37 μmol/L) and PNA-FITC (1 ng/L) and flow cytometry. Chemicals were purchased from Sigma (Madrid, Spain). Part of the sample was divided into six aliquots and diluted 1:1 with different extenders: UL4: Tes-Tris-Fructose (TTF), 10% egg yolk (EG), and 4% glycerol (G); UL8: TTF, 20% EG, and 8% G; AND4: Andromed® (Minitüb, Tiefenbach, Germany) and 4% G; AND7: Andromed® and 7% G; GENT: Gent 1045; and INRA: INRA96 and 4% G. Andromed, Gent, and INRA are commercial extenders. Samples were cooled to 5°C (−0.2°C/min) and then diluted to 200 × 106 sperm/mL. Samples were packed (0.5-mL straws) and frozen using a biofreezer (from 5°C to −15°C at −15°C/min, and from −15°C to −100°C at −25°C/min). Samples were thawed at 65°C for 6 s, and assessed as for pre-freezing (Table 1). Post-thawing motility recovery using AND7 was excellent. The highest viability recovery was achieved by UL4, although that in AND7 was similar. The poor results of equine commercial extender Gent 1045 in this species are remarkable. Our results highlight the importance of species differences in the field of sperm cryopreservation. It is necessary to carry out continuous research for optimizing cryopreservation protocols in order to create germplasm banks for wild species. Table 1. Quality assessment results

9 BIRTH OF CALVES AFTER ARTIFICIAL INSEMINATION WITH CRYOPRESERVED BOVINE EPIDIDYMAL SPERMATOZOA HARVESTED FROM POSTMORTEM BULLS

2009 ◽  
Vol 21 (1) ◽  
pp. 105 ◽  
Author(s):  
C. A. Guerrero ◽  
G. Gentry ◽  
J. Saenz ◽  
K. R. Bondioli ◽  
R. A. Godke
Keyword(s):  
Sperm Concentration ◽  
Egg Yolk ◽  
Beef Cows ◽  
Gestation Length ◽  
Epididymal Sperm ◽  
Average Birth Weight ◽  
Male Gametes ◽  
Potential Source ◽  
Mixed Breed ◽  
The Mean

Recently, interest in the preservation of epididymal sperm as a potential source of valuable genes for genome resource banks has increased. The ability to recover and cryopreserve postmortem epididymal sperm will allow the use of valuable gametes from a breeding male that dies unexpectedly. The objective of this study was to produce pregnancies and live births from AI by using frozen–thawed bovine caudal epididymal sperm. Paired testes were obtained from mature, mixed-breed beef bulls (n = 3) from a local abattoir and transported to the laboratory (within 5 h postmortem) in a Styrofoam box at a temperature that ranged between 28 and 29°C. The mean ± SEM weights of the pair of testes and caudae epididymides were 345 ± 9g and 10 ± 2g, respectively. Epididymal sperm from each bull were harvested by multiple incisions from the caudae epididymides, pooled, and then rinsed with 4 mL of egg yolk Tris-glucose citric acid monohydrate extender (EYT-GC). Harvested sperm samples were placed in a refrigerator at 4°C for 4 h before the addition of cryoprotectant. Samples were diluted slowly over a period of 30 min 1:1 in EYT-GC extender containing 14% glycerol to obtain a final concentration of 7% glycerol. Sperm concentration was adjusted to 35 million/straw and loaded into previously cooled 0.5-mL plastic straws. Straws were placed 2 cm above liquid nitrogen (LN2) vapors for 10 min and then plunged into LN2. Sperm were stored for 1 year before thawing. Luteal-phase crossbred beef cows were synchronized with a single 25-mg injection (i.m.) of prostaglandin, and those displaying standing estrus (n = 6) were each inseminated with 1 straw by a single technician from 1 bull 12 to 18 h after the onset of standing estrus. Straws were thawed for AI in a 37°C water bath for 40 s. Three of the 6 cows inseminated (50%) were diagnosed as pregnant by transrectal ultrasonography at 45 days post-AI. All 3 pregnant cows (100%) delivered healthy singleton calves (2 males and 1 female, with an average birth weight of 37 ± 2.5 kg), resulting in a mean gestation length of 286 ± 1.9 days (range: 282–288 days). We can conclude that epididymal sperm can be extracted by 5 h postmortem from bull testes, frozen, and subsequently used for the production of live offspring from AI. Further research is needed to improve this technology to optimize the utilization of valuable bovine male gametes.

scholarly journals Outlining adequate protocols for Lidia bull epididymal storage and sperm cryopreservation: use of glycerol, dimethylformamide and N-acetylcysteine

2017 ◽  
Vol 15 (3) ◽  
pp. e0405
Author(s):  
Elvira Matilla ◽  
Lauro González-Fernández ◽  
Felipe Martínez-Pastor ◽  
Nuria Hernández ◽  
Carolina Tobajas ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Sperm Motility ◽  
Mitochondrial Membrane ◽  
Egg Yolk ◽  
Ros Production ◽  
Epididymal Sperm ◽  
Sperm Cryopreservation ◽  
Cattle Industry ◽  
Bull Sperm

The Lidia bovine breed is an important hallmark of the Spanish cattle industry. Bulls are selected based upon aggressiveness and epididymal sperm cryopreservation is the way to obtain and store their genetics. There are not specifically designed protocols yet to perform Lidia bull sperm cryopreservation. The present study aimed to determine if a tris-fructose-citrate-egg yolk (20% v/v; TFY) extender supplemented with 7% glycerol (TFY1) or 3.5% glycerol plus 3.5% dimethylformamide (DMF; TFY2) are suitable media for cryopreservation of epididymal Lidia bull sperm. Moreover, the effect of N-acetylcysteine (NAC), a potent antioxidant, was evaluated. The epididymis were stored at 4°C for 24, 48, 72 or 96 h, and both freezing media were tested as such or supplemented with 1 or 2.5 mM of NAC. Our data demonstrated that post-thaw viability was well maintained (TFY1: 50.8% ± 1.9 at 24 h and 52.4% ± 0.8 at 96 h and TFY2: 52.6% ± 1.6 at 24 h and 56.1% ± 1.8 at 96 h; mean % ± SEM; p>0.05) as also were total and progressive sperm motility, high mitochondrial membrane potential, ROS production, DNA status and acrosomal intactness of Lidia bull sperm up to 96 h of epididymal storage, all extender variations being similar (p>0.05). In conclusion, the use of TFY medium supplemented either with 7% glycerol alone or the combination of 3.5% glycerol and 3.5% DMF were equally safe choices for epididymal Lidia bull sperm cryopreservation, and NAC addition did not significantly improve sperm post-thaw quality.

Cryopreservation of Spix's yellow-toothed cavy epididymal sperm using Tris- and coconut water-based extenders supplemented with egg yolk or Aloe vera

Cryobiology ◽  
2021 ◽  
Author(s):  
Samara Sandy Jeronimo Moreira ◽  
Andreia Maria da Silva ◽  
Ana Liza Paz Souza ◽  
Erica Camila Gurgel Praxedes ◽  
João Batista Freire de Souza Junior ◽  
...  
Keyword(s):  
Aloe Vera ◽  
Egg Yolk ◽  
Coconut Water ◽  
Epididymal Sperm ◽  
Water Based

scholarly journals Effect of age and season on the Epididymal Sperm and testosterone level in camel (camelus dromedarius)

2014 ◽  
Vol 38 (1) ◽  
pp. 24-29
Author(s):  
Ali A. Abd
Keyword(s):  
Testosterone Level ◽  
Sperm Concentration ◽  
Serum Testosterone ◽  
Cold Season ◽  
Serum Testosterone Level ◽  
Epididymal Sperm ◽  
Hot Season ◽  
Live Sperm ◽  
Effect Of Age

The aim of this study was to evaluate the effect of age and season on the epididymal sperm and level of testosterone in camel. A total 103 camel testes samples were collected from Al-Najaf slaughter house during a cold season (December 2012, January and February 2013) and moderatehot season (March, April and May, 2013). Testes were divided into 3 Gs according to camel age, G1 included the testes of 2-3years, G 2 (3 – 4years) and over 4 years (G 3). Blood samples were collected for determination of serum testosterone level. The sperms were obtained from the tail of epididymis from all animals groups and the results of the sperms individual motility percentage was increased at the level of (P< 0.05) significantly with age progress in both seasons. Also, sperm motility of G3 recorded a significantly higher than those of G1 and G2 in cold and moderate-hot seasons .The live percentage of G 3 animals was 90.01% in cold season with a significantly higher than those of other Gs and in both seasons. However, the live sperm percentage of G 3 during moderate-hot season was 87.82% and G 2 during moderate-hot and cold seasons were 88.58 and 88.72% respectively, showed significantly higher than those of G1 during cold and moderate-hot seasons .The concentration of sperm obtained from epididymis tail of bulls camel significantly increase with age progress in both seasons. However, the mean of the sperm concentration in cold season showed significance higher than those in moderate-hot season in animals of G 1 and 3, respectively. The abnormal morphologically sperm percentage of animals G 1 in both cold and moderate-hot seasons were significantly higher than those of G3.The testosterone levels of the young animals (under 4 years) increased gradually and reached its peak in February 2.28 ng/ml and March 2.27ng/ml. In the same trend older animal (more than 4 years) was showed 8.14 and 7.35 ng/ml, respectively. The older animals showed a significantly monthly, higher values than those of the younger animals in their testosterone level started from January up to May. In conclusions during cold months the camel over 4 years shows higher percentage of epididymal sperms parameters (live and individual motility) and sperms concentration as well as serum testosterone level.

Cryopreservation of collared peccary (Pecari tajacu L., 1758) epididymal sperm using extenders based on Tris and powdered coconut water (ACP®-116c)

Zygote ◽  
2018 ◽  
Vol 26 (4) ◽  
pp. 301-307 ◽  
Author(s):  
José A. B. Bezerra ◽  
Andréia M. Silva ◽  
Patrícia C Sousa ◽  
Lívia B. Campos ◽  
Érica C. G. Praxedes ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Coconut Water ◽  
Computer Assisted ◽  
Epididymal Sperm ◽  
Sperm Plasma Membrane ◽  
Pecari Tajacu ◽  
Functional Membrane

SummaryThe aim of this study was to establish a functional freezing–thawing protocol for epididymal sperm of collared peccaries (Pecari tajacu L., 1758) by comparing different extenders. The epididymal sperm from 12 sexually mature males was recovered by retrograde flushing using Tris-based or coconut water-based (ACP®-116c) extenders. After initial evaluation, samples were diluted and frozen with the same extenders to which 20% egg yolk and 6% glycerol were added. After 2 weeks, thawing was performed at 37°C/60 s and sperm motility, vigour, morphology, functional membrane integrity, sperm viability, sperm plasma membrane integrity, and a computer-assisted semen analysis (CASA) were assessed. In addition, to evaluate the survival of frozen–thawed sperm, a thermal resistance test (TRT) was executed. Samples preserved using Tris were in better condition compared with those preserved using ACP®, showing higher values for most assessments performed, including CASA and the TRT (P<0.05). After determining Tris to be the better of the two extenders, additional samples were thawed using different thawing rates (37°C/60 s, 55°C/7 s, 70°C/8 s). Sperm thawed at 37°C/60 s had the greatest preservation (P<0.05) of viability (54.1 ± 5.9%) and functional membrane integrity (43.2 ± 5.4%), and had higher values for various CASA parameters. In conclusion, we suggest the use of a Tris-based extender added to egg yolk and glycerol for the cryopreservation of epididymal sperm obtained from collared peccaries. In order to achieve better post-thawing sperm quality, we suggest that samples should be thawed at 37°C/60 s.

Epididymal sperm from Spix ’s yellow-toothed cavies sperm successfully cryopreserved in Tris extender with 6% glycerol and 20% egg yolk

2018 ◽  
Vol 191 ◽  
pp. 64-69
Author(s):  
Andréia M. Silva ◽  
Erica C.G. Praxedes ◽  
Lívia B. Campos ◽  
Luana G.P. Bezerra ◽  
Samara S.J. Moreira ◽  
...  
Keyword(s):  
Egg Yolk ◽  
Epididymal Sperm

54 CRYOPRESERVATION OF DOMESTIC CAT EPIDIDYMAL SPERM IN A DEFINED EXTENDER WITHOUT ANIMAL OR PLANT PROTEINS

2012 ◽  
Vol 24 (1) ◽  
pp. 139
Author(s):  
J. R. Saenz ◽  
C. Dumas ◽  
B. L. Dresser ◽  
M. C. Gómez ◽  
R. A. Godke ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Egg Yolk ◽  
Domestic Cat ◽  
The Other ◽  
Initial Assessment ◽  
Computer Assisted ◽  
Plant Proteins ◽  
Epididymal Sperm ◽  
Sperm Suspension ◽  
Sperm Survival

Previously, we have shown that survival of cat sperm is maintained in both non-egg yolk, semi-defined extenders and in extenders with greatly reduced levels of egg yolk (2%). Usually, cryoprotectant is added to extended samples after gradual cooling to 4°C, but recent reports have shown that satisfactory sperm survival can be obtained after addition at 22°C. Here, our objectives were to examine sperm survival after (1) cryopreservation from 22°C vs after gradually cooling to 4°C or (2) cryopreservation in a completely defined extender without animal or plant proteins vs extender + 2% egg yolk. Epididymides from local veterinary clinics were dissected in HEPES 199 medium (He199). The sperm suspension was filtered (40 μ), layered onto a density gradient column and centrifuged at 650 × g for 20 min. Then, the sperm pellet was resuspended in 1 mL of He199 and centrifuged for 5 min at 800 × g and the subsequent pellet was extended in TEST Buffer with either 0% (0% EY) or 2% egg yolk (2% EY). Next, 0% EY samples were further split into 2 groups—either gradually cooled to 4°C before 12% glycerol (1:1) was added (4C-0%EY) or 12% glycerol (1:1) was added at 22°C without cooling (22C-0%EY). Control samples extended in 2% EY were cooled to 4°C before addition of 12% glycerol (1:1) (4C-2%EY). Samples were loaded into 0.25-mL straws and placed in a –80°C freezer for 20 min before storage in LN2. Sperm samples were thawed in air (22°C) for 5 s and immersed in a 60°C water bath for 5 s. After a 7-step addition of He199, samples were centrifuged at 800 × g for 5 min and pellets resuspended in He199. Sperm samples were evaluated for motility (Mot; computer-assisted semen analysis, 37°C) at 0 h (initial assessment), after cooling to 4°C (PC) and at 0-h (0-PT) and 3-h post-thaw (3-PT) incubation at 37°C. Membrane integrity (MI; SYBR 14-PI) and acrosomal status (AS; FITC-PNA) were analysed at the initial assessment, 0-PT and 3-PT. Results are shown in Table 1. At 4°C (PC), sperm extended in 0% EY and 2% EY maintained 92 and 91%, respectively, of their initial motility (66%). At 0-PT and 3-PT, motility in the 3 groups had decreased by >50% and >70%, respectively. Motility at 3-PT in the 22C-0%EY treatment was less than the other 2 treatments (P < 0.05; 1-way ANOVA). At 0-PT, samples in the 4C-2%EY group had a higher membrane integrity value (P < 0.05) than did the 22C-0%EY group, whereas that of the 4C-0%EY group was not different from the other 2 groups. However, at 3-PT, both groups cooled to 4°C before cryopreservation had higher membrane integrity values (P < 0.05) than the group cryopreserved at 22°C. At 0-PT and 3-PT, the percentage of sperm with intact acrosomes ranged from 69% (4C-2%EY) to 59% (22C-0%EY) and from 55% (4C-2%EY) to 43% (22C-0%EY) of the initial value (89%), respectively. In summary, we demonstrated that cat epididymal sperm could be frozen successfully in a completely defined TEST-buffered extender. Furthermore, we confirmed that addition of cryoprotectant (i.e. glycerol) after gradual cooling to 4°C is beneficial to post-thaw survival. Table 1.Motility (Mot), membrane integrity (MI) and acrosomal status (AS) of cat epididymal sperm before and after cryostorage

35 EFFECTS OF THAWING TEMPERATURE OF FROZEN SEMEN ON VIABILITY OF REFROZEN AND THAWED CHICKSO (KOREAN BRINDLE CATTLE) AND KOREAN ALBINO CATTLE SPERMATOZOA

2016 ◽  
Vol 28 (2) ◽  
pp. 147
Author(s):  
S. W. Kim ◽  
C. Y. Choe ◽  
D. K. Kim ◽  
A. R. Choi ◽  
H. H. Seong
Keyword(s):  
Genetic Resources ◽  
Sperm Concentration ◽  
Cooling Rates ◽  
Mean Values ◽  
Freeze Thaw ◽  
Frozen Semen ◽  
Native Cattle ◽  
Viable Sperm ◽  
The Mean ◽  
Thawing Procedure

Germplasm cryopreservation from a desired species with agricultural and genetic importance would protect them from the risk for extinction. Semen freezing from Korean native cattle would be a good approach for protecting genetic resources due to their limited numbers. It has been known that sperm could resist cryo-damages by freeze-thaw cycles. Thus, we performed 2 refreezing experiments with different initial thawing temperatures using frozen Korean native cattle semen. A total of 5 Hanwoo, Korean Albino, and brindle cattle were used as semen donors. After thawing by using 5°C/2 min or 37°C/40 s with cooling rates, the semen was diluted with the same volume of cryo-media in the first thawing temperature and refrozen. Sperm motilities were determined and compared between animals and groups after rethawing. The mean sperm concentration and motility was 45 × 106 mL–1 (range 2.3 to 89 × 106 mL–1) and 40% (range 13 to 55%). Mean values of motility and viability of sperm that underwent second preservation were significantly higher in 5°C than in 37°C (P < 0.01). However, the activity of viable sperm thawed at 5°C was significantly decreased before refreezing. It is estimated that refreezing of frozen semen from rare Korean native cattle is possible with resistant properties of survived spermatozoa. The higher motility and viability of refrozen semen could be obtained with 5°C thawing procedure for reuse of frozen semen.

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