scholarly journals Optimalisasi Pembekuan Sperma Limbah Kauda Epididimis Kambing Lokal dengan Metode Bertahap dan Stabilisasi

2018 ◽  
Vol 35 (2) ◽  
pp. 150
Author(s):  
Naela Wanda Yusria Dalimunthe ◽  
M. Rosyid Ridlo ◽  
Agung Budiyanto
Keyword(s):  
Liquid Nitrogen ◽  
Multistep Methods ◽  
Egg Yolk ◽  
Single Step ◽  
Dilution Method ◽  
Sperm Suspension ◽  
Sperm Utilization ◽  
One Way Anova ◽  
Test Correlation

Buck slaugthering produce waste such as testicles including epididymis which contain fertile sperm. Utilization of cauda epididymis as the sources of sperm for producing goat frozen sperm was not reported yet. The aims of this study were improving the frozen-thawed sperm using stabilization and multistep methods which recovered from the waste of buck slaughtering as the source of sperma. Cauda epididymis spermatozoa which was washed then diluted using extender 1 (Tris-citrate-antibiotics) and extender 2 (extender 1- glycerol-egg yolk). The extender 2 addition was performed by single or multistep methods then freezed. Modification in the pre freezing proces were performed by comparing the conventional equilibration and stabilization methods. The sperm suspension was incubated in 4°C for 2 hours after filling-sealing into straws on the equilibration group whether the stabilization group was cooled in tube 15 mL. All cooled straws from both groups were placed 4 cm horizontally on liquid nitrogen surface for 10 minutes and then plunged into liquid nitrogen for storage. The evaluation of motility parameters such as pattern of the movement and motility percentation were done followed the standard methodology. The student t-test, correlation and one-way ANOVA were used for data analysis with P<0.05. The results showed that multistep dilution method could increase the motility (25.0 ± 1.8 %) compared with single step (18.3 ± 1.7 %). Pre freezing method with stabilization also resulted higher motility (24.2 ± 2.0 %) than equilibration method (17.5 ± 2.8 %). The pattern of the movement were not different between all methods and its combination. The multistep dilution method and stabilization cooling method as well as its combination seems could increase the quality of frozen-thawed cauda epididymis spermatpzoa of local buck.

scholarly journals Cryopreservation of Indonesian native chicken semen by using dimethyl sulfoxide and various level of ethylene glycol as cryoprotectants

2020 ◽  
Vol 21 (12) ◽  
Author(s):  
KHAERUDDIN KHAERUDDIN ◽  
JUNAEDI JUNAEDI ◽  
HASTUTI HASTUTI
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Egg Yolk ◽  
Sperm Cryopreservation ◽  
Randomized Design ◽  
Native Chicken ◽  
Ringer’S Lactate ◽  
Completely Randomized Design

Abstract. Khaeruddin, Junaedi, Hastuti. 2020. Cryopreservation of Indonesian native chicken semen by using dimethyl sulfoxide and various level of ethylene glycol as cryoprotectants. Biodiversitas 21: 5718-5722. Imported purebred chickens are becoming more popular and a regular staple in Indonesia. Therefore, it is necessary to strengthen conservation efforts to preserve Indonesian chickens, one of which is by means of sperm cryopreservation. This study aimed to determine the effects of the addition of DMSO and different concentrations of ethylene glycol to a Ringer’s lactate egg yolk (RLY)-or coconut water egg yolk (CWY)-based extender on the quality of frozen-thawed Indonesian chicken sperm. This study was used nine Indonesian native roosters about 20 months of age. The semen extenders used in this study were RLY + DMSO 7%, RLY + ethylene glycol 3%, RLY + ethylene glycol 5%, RLY + ethylene glycol 7%, CWY + DMSO 7%, CWY + ethylene glycol 3%, CWY + ethylene glycol 5% and CWY + ethylene glycol 7%. Liquid semen was packaged in 0.25 mL straw, then cooled at 5oC for 2 hours, frozen at 5 cm above liquid nitrogen for 10 minutes, following stored in a liquid nitrogen container for 24 hours. The semen straws were thawed at 37oC for 30 seconds. Statistical analysis for multiple comparisons was performed as a completely randomized design with eight treatment levels and seven replications. The results showed that there were no differences in sperm motility, recovery rate, and abnormality between extenders after the freeze-thaw process. Whereas, RLY + DMSO 7% was the highest sperm viability.

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

scholarly journals CRYOPRESERVATION OF KAMPUNG ROOSTER SEMEN USING EGG YOLK DILUENT FROM FOUR TYPES OF POULTRY WITH DIFFERENT CONCENTRATIONS

2019 ◽  
Vol 13 (3) ◽  
Author(s):  
Khairuddin Khairuddin ◽  
Muhammad Erik Kurniawan ◽  
Soman Soman
Keyword(s):  
Liquid Nitrogen ◽  
Recovery Rate ◽  
Egg Yolk ◽  
Spermatozoa Motility ◽  
Chicken Egg ◽  
Nitrogen Vapor ◽  
Before And After ◽  
Egg Yolks ◽  
Chicken Egg Yolk

The aim of this study was to determine the type and the best concentration of egg yolk in maintaining the quality of kampung rooster spermatozoa during cryopreservation. This study used a completely randomized factorial pattern design with the first factor was the type of egg yolk (purebred chicken, kampung chicken, duck, and quail) and the second factor was the concentration of egg yolk (5%, 10%, and 15%). Semen was collected from twelve kampung roosters using massage method. Immediately after collection, the semen was evaluated macroscopically and microscopically. Semen with more than 70% motility was used in this study. The semen was diluted, packed in a ministraw, equilibrated, and frozen using liquid nitrogen vapor and stored in a liquid nitrogen container for 24 hours. Observation of spermatozoa motility was carried out in fresh semen, diluted semen, after equilibration and after thawing with four replications. The results showed that the type of egg yolk treatment had no effect (P0.05) on the recovery rate and motility of spermatozoa before and after cryopreservation, but egg yolk concentration had a highly significant effect (P0.01) on the quality of spermatozoa. Egg yolks in 10-15% concentration had spermatozoa motility and recovery rate higher than egg yolk with 5% concentration. In conclusion, purebred chicken egg yolk, kampung chicken egg yolk, duck egg yolk, and quail egg yolk each in diluent can be used to maintain the quality of kampung rooster spermatozoa at a concentration of 10-15% during cryopreservation.

Erratum to: 56 SINGLE LAYER CENTRIFUGATION THROUGH PURESPERM® 80 IMPROVES QUALITY OF CRYOPRESERVED DOG SPERMATOZOA

2013 ◽  
Vol 25 (1) ◽  
pp. 175
Author(s):  
L. Alcaráz ◽  
M. Hidalgo ◽  
M. J. Galvez ◽  
D. Acha ◽  
I. Ortiz ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Semen Quality ◽  
Semen Analysis ◽  
Gradient Centrifugation ◽  
Single Layer ◽  
Egg Yolk ◽  
Final Concentration ◽  
Viable Sperm

Density gradient centrifugation with PureSperm® (PureSperm® 40 + PureSperm® 80; Nidacon International, Mölndal, Sweden) has been satisfactorily used to enhance the quality of dog semen samples; however, no studies have been performed on the effect of single layer centrifugation (SLC) with PureSperm® on frozen–thawed dog semen. The aim of this study was to investigate if SLC with PureSperm® 80 can improve the post-thaw semen quality of dog. Semen from 5 dogs was collected by digital manipulation. Two ejaculates from each dog were centrifuged with Tris-based extender, supernatant was removed, and sperm pellet was suspended to a final concentration of 300–400 × 106 sperm mL–1 with CaniPROTM Freeze A plus 20% egg yolk at 22°C. Extended semen was cooled to 5°C within an hour and then diluted to a final concentration of 150–200 × 106 sperm mL–1 in CaniPROTM Freeze B plus 20% egg yolk at 5°C, loaded in 0.5-mL plastic straws and frozen horizontally in ranks placed 4 cm above the surface of liquid nitrogen vapors for 10 min, after which they were directly placed in liquid nitrogen. After 24 to 48 h of storage, straws were thawed in a water bath at 37°C for 30 s. After thawing, semen samples were divided in 2 aliquots: one of them was used as control and the other one was processed by SLC PureSperm® 80. Assessment of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining), and viability [triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123] were evaluated in control and treated semen samples. Data were studied by ANOVA. Results are expressed as mean ± SEM. Significant (P < 0.001) differences were found between SLC-treated and control semen for sperm motility (percentage of total motile spermatozoa: 93.65 ± 0.05 v. 83.79 ± 0.13; percentage of progressive motile spermatozoa: 79.38 ± 6.66 v. 54.61 ± 16.11), morphology (86.45 ± 0.01 v. 83.51 ± 0.01), and viability (percentage of viable sperm with an intact acrosome: 58.32 ± 0.04 v. 36.50 ± 0.17; percentage of viable sperm with an acrosome reaction: 2.81 ± 0.01 v. 9.74 ± 0.21). Based on our results, we can conclude that SLC with PureSperm® 80 is an alternative and successful method for improving the quality of frozen–thawed dog spermatozoa, selecting good-quality spermatozoa (motile, morphologically normal, viable, and acrosome intact spermatozoa) from the rest of the semen sample.

scholarly journals Erratum to: 56 SINGLE LAYER CENTRIFUGATION THROUGH PURESPERM® 80 IMPROVES QUALITY OF CRYOPRESERVED DOG SPERMATOZOA

2013 ◽  
Vol 25 (3) ◽  
pp. 587
Author(s):  
L. Alcaráz ◽  
M. Hidalgo ◽  
M. J. Galvez ◽  
D. Acha ◽  
I. Ortiz ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Semen Quality ◽  
Semen Analysis ◽  
Gradient Centrifugation ◽  
Single Layer ◽  
Egg Yolk ◽  
Final Concentration ◽  
Viable Sperm

Density gradient centrifugation with PureSperm® (PureSperm® 40+PureSperm® 80; Nidacon International, Mölndal, Sweden) has been satisfactorily used to enhance the quality of dog semen samples; however, no studies have been performed on the effect of single layer centrifugation (SLC) with PureSperm® on frozen–thawed dog semen. The aim of this study was to investigate if SLC with PureSperm® 80 can improve the post-thaw semen quality of dog. Semen from 5 dogs was collected by digital manipulation. Two ejaculates from each dog were centrifuged with Tris-based extender, supernatant was removed, and sperm pellet was suspended to a final concentration of 300–400×106spermmL–1 with CaniPROTM Freeze A plus 20% egg yolk at 22°C. Extended semen was cooled to 5°C within an hour and then diluted to a final concentration of 150–200×106spermmL–1 in CaniPROTM Freeze B plus 20% egg yolk at 5°C, loaded in 0.5-mL plastic straws and frozen horizontally in ranks placed 4cm above the surface of liquid nitrogen vapors for 10min, after which they were directly placed in liquid nitrogen. After 24 to 48h of storage, straws were thawed in a water bath at 37°C for 30s. After thawing, semen samples were divided in 2 aliquots: one of them was used as control and the other one was processed by SLC PureSperm® 80. Assessment of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining), and viability [triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123] were evaluated in control and treated semen samples. Data were studied by ANOVA. Results are expressed as mean ± SEM. Significant (P&lt;0.001) differences were found between SLC-treated and control semen for sperm motility (percentage of total motile spermatozoa: 93.65±0.05 v. 83.79±0.13; percentage of progressive motile spermatozoa: 79.38±6.66 v. 54.61±16.11), morphology (86.45±0.01 v. 83.51±0.01), and viability (percentage of viable sperm with an intact acrosome: 58.32±0.04 v. 36.50±0.17; percentage of viable sperm with an acrosome reaction: 2.81±0.01 v. 9.74±0.21). Based on our results, we can conclude that SLC with PureSperm® 80 is an alternative and successful method for improving the quality of frozen–thawed dog spermatozoa, selecting good-quality spermatozoa (motile, morphologically normal, viable, and acrosome intact spermatozoa) from the rest of the semen sample.

scholarly journals Antioxidant Potential of Ferulic Acid on the Freezability of Bull Semen

2019 ◽  
Vol 4 (3) ◽  
pp. 1-4
Author(s):  
Omur AD
Keyword(s):  
Ferulic Acid ◽  
Liquid Nitrogen ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Single Step ◽  
Spermatozoa Motility ◽  
Bull Semen ◽  
Sperm Density ◽  
Significant Difference ◽  
Artificial Vagina

Ejaculates were collected twice a week from the bulls, via an artificial vagina, during two weeks. The suitable ejaculates obtained for sperm density (≥ 1.4 × 10 9 spermatoz oa / ml) and for motility (≥ 75%) were used for dilution and freezing of semen. A Tris - based extender (Tris 297.58mM, citric acid 96.32mM, fructose 82.66mM, egg yolk 15% (v/v), glycerol 5% (v/v), gentamicin 0.1 ml / 100ml, pH 6.8 - 7.0) was used as the base extender (cryopreservation diluent). Pooled ejaculate was split into 2 equal aliquots and diluted at 32 °C with base extender containing ferulic acid (100 μM) and no antioxidant (control), respectively. Each aliquot was diluted to a final semen concentrati on of approximately 1.2 × 10 8 sperm/ml (single step dilution), in 15 - ml polypropylene centrifuge tubes. After dilution, semen samples were kept at room temperature for 10 minutes then, the diluted semen samples were aspirated into 0.25 ml French straws, seal ed with polyvinyl alcohol powder and equilibrated at 5 °C for 3 h. After equilibration, the straws were frozen in liquid nitrogen vapour (4 cm above the liquid nitrogen, ~ - 100 o C ) for 10 min and then plunged into liquid nitrogen for storage, - 196 o C . In the study, sperm samples containing antioxidant and non - antioxidant were evaluated for spermatozoa motility and membrane integrity after freezing / thawing. In the present study, no statistically significant difference was found between the control and experim ental groups for motility and membrane integrity after freeze - thawing. The application consisted of 4 replications.

scholarly journals Kualitas Sperma Beku Sapi Bali dalam Pengencer Air Kelapa Modifikasi dengan Berbagai Aras Dimethyl Sulfoxide (FROZEN SPERM QUALITY OF BALI BULLS IN MODIFIED COCONUT WATER EXTENDER WITH DIFFERENT DIMETHYL SULFOXIDE CONCENTRATION)

2019 ◽  
Vol 20 (1) ◽  
pp. 93
Author(s):  
Thomas Mata Hine ◽  
Kirenius Uly ◽  
Wilmientje Marlene Nalley ◽  
Heri Armadianto
Keyword(s):  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Optimal Concentration ◽  
Coconut Water ◽  
Low Molecular Weight ◽  
Frozen Semen ◽  
Artificial Vagina

Dimethyl Sulfoxide (DMSO) is one type of cryoprotectant which has a low molecular weight so that it is easier to enter cells when cryopreservation. The purpose of this study was to explore the optimal concentration of DMSO in modified coconut water (mCW) extender that were able to maintain frozen sperm quality of bali bulls. Semen was collected from two four-year old bali bulls by artificial vagina. Good quality semen diluted with mCW (young coconut water + 20% egg yolk + 7.5 % moringa leaf extract) and supplemented by 3, 5, or 7% DMSO. Semen was filled into 0.25 ml ministraw, and was incubated in a refrigerator at 5°C for four hours, frozen on the surface of liquid nitrogen for 10 minutes and then dipped into liquid nitrogen. The quality of post thawing sperm was measured 24 hours later by placing the ministraw of frozen semen into water at 37oC for 30 seconds. Data were analyzed by analysis of variance and continued with Duncan test. Postthawing observations showed that bali bulls sperm cryopreserved at 3% DMSO yielded higher motility and viability (p<0.05) i.e. 36 and 44.15%, than DMSO 5% i.e. 18 and 23.65%, and DMSO 7% i.e. 7 and 12.62%. The recovery rate of sperm cryopreserved at 3% DMSO was also higher (p<0.05) than DMSO 5 and 7%, successively 45.65, 23.06, and 8.86%. The results of this study concluded that the optimal concentration of DMSO in mCW diluent to maintain frozen sperm quality of bali bulls was 3%. 

scholarly journals Ram and Goat Semen Immunosexed and Diluted in Powdered Coconut Water-Based Preservation Medium (ACP101/102c).

2021 ◽  
Vol 49 ◽  
Author(s):  
Bruna Farias Brito ◽  
Bárbara Mara Bandeira Santos ◽  
Leonardo Alves Rodrigues Cabral ◽  
Luiz Carlos Pinheiro Maia ◽  
Natanael Aguiar Braga Negreiros ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Specific Protein ◽  
Coconut Water ◽  
In Vivo Studies

Background: Sperm sexing aims to separate sperm populations in carriers of the “X” or “Y” chromosome. Currently, flow cytometry is a technique that allows greater accuracy; however, it causes structural changes in sperm, reduces viability, and has a high cost. As a result, other methods have been researched, including immunosexing, which uses monoclonal antibodies to detect sex-specific surface antigens. Thus, the objective of this study was to evaluate the immunosexing technique using a monoclonal antibody against sex-specific protein (HY) in the conservation of ram and goat semen in ACP101/102c.Materials, Methods & Results: Ejaculates from 5 rams and 5 goats were collected with the aid of an artificial vagina; they were evaluated and submitted to the immunosexing protocol, according to the manufacturer's recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with “Y” chromosomes (HY; HY Biotechnology, Rio de Janeiro, RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP ram (ACP101/102c + 20% egg yolk + 7% glycerol) and ACP goat (ACP101/102c + 2.5% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated at 4°C, stabilized for 30 min, frozen in liquid nitrogen vapor (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were evaluated in natura (T1), after immunosexing (T2) and after thawing (T3) for sperm motility subjectively using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and morphologically defect spermatozoa were determined. In the evaluation of ram semen regarding sperm motility and IMP, no statistically significant differences were observed between treatments after sexing in the evaluation of absolute data (P > 0.05), with the difference being observed only between T1 and T2, and T3 (P < 0.05). Regarding the relative percentage and sperm morphology, no statistically significant differences were observed (P > 0.05). Regarding the evaluation of goat semen samples, the motility parameters were consistent with the technique submitted; however, the IMP data did not appear as expected, requiring further evaluation for a better assessment of the technique for this species.Discussion: The data obtained from ram semen submitted to the immunosexing protocol, regarding the absolute evaluation of motility and IMP, demonstrated that the non-sexed semen (T1) was superior to the sexed treatments (T2 and T3); however, it is noteworthy that freezing started with approximately 50% of the cells, since the immunosexing technique results in a loss of viability of approximately 50% of the sperm, which corresponds to the ratio of sperm carrying the X chromosome. In addition, when the data in this study were transformed into relative values, no statistical differences were observed, indicating that the immunosexing protocol, as well as the freezing protocol, did not significantly affect the quality of ram sperm cells. In relation to the immunosexing of goat semen, future studies should be conducted in vitro to define a more appropriate protocol for the species and, in addition, in vivo studies should be performed to prove the quality of the technique. It was concluded that the immunosexing process using a monoclonal antibody against sex-specific protein (HY) associated with the use of powdered coconut water diluent (ACP101/102c) in the cryopreservation of semen proved to be efficient in the in vitro evaluation of ovine species.

scholarly journals THE ROLE OF GUAVA FRUIT FILTRATE ON MAINTAINING THE PRE-FREEZING AND POST-THAWED QUALITY OF BALI BULL SPERMATOZOA

2018 ◽  
Vol 12 (2) ◽  
Author(s):  
I Wayan Lanus Sumadiasa ◽  
Lukman Lukman ◽  
Rodiah Rodiah
Keyword(s):  
Liquid Phase ◽  
Light Microscopy ◽  
Liquid Nitrogen ◽  
Egg Yolk ◽  
Bovine Semen ◽  
Guava Fruit ◽  
Artificial Vagina

This study aimed to determine the role of guava fruit filtrate (GFF) on maintaining the pre-freezing and post-thawed quality of Bali bullspermatozoa in tris-egg yolk (TEY) extender. The bovine semen was collected using artificial vagina twice a week (n= 10). Ejaculate sampleswere divided into four tubes and each tube was respectively added the following diluents: TEY, TEY + 5% GFF, TEY + 10% GFF, and TEY +15% GFF. The diluted semen was filled into the straws (0.25 mL) and cooled in refrigerator at 4° C for 2.5 hours. The samples were kept onvapor phase of liquid nitrogen (N2) (-120° C) for 10 minutes prior to be stored in liquid phase (-196° C). The quality of pre-freezing and postthawedspermatozoa in straw sample (i.e. motility, viability, and abnormality) was evaluated under light microscopy at 400x magnification. Datawere analysed statistically. The percentage of motility and viability both on pre-freezing and post-thawed process were significantly higher insemen diluted with TEY + 10% GFF compared to control (TEY), TEY + 5% GFF, and TEY + 15% GFF. Addition of 10% GFF into tris-egg yolkextender play role for maintaining the quality of pre-freezing and post-thawed Bali bull spermatozoa.

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