37 DOUBLE FREEZING AND THAWING OF NGUNI BULL SEMEN

2016 ◽  
Vol 28 (2) ◽  
pp. 148
Author(s):  
M. L. Mphaphathi ◽  
M. M. Seshoka ◽  
T. R. Netshirovha ◽  
Z. C. Raphalalani ◽  
T. C. Chokoe ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Egg Yolk ◽  
Freezing And Thawing ◽  
Sperm Analysis ◽  
Treatment Groups ◽  
Bull Semen ◽  
Before And After ◽  
Repeated Freezing ◽  
Thawing Process ◽  
Marked Decline

Indigenous bulls semen are important for conservation programs. The objectives of this study were to evaluate the effects of repeated freezing and thawing on sperm motility characteristics. Semen was collected from 4 Nguni bulls by means of electro ejaculator and stored in a thermo flask (37°C). Sperm total motility, progressive and nonprogressive motility, and velocity were assessed using computer-aided sperm analysis before and after freezing. Semen was then diluted with egg yolk citrate extender (fraction A), then followed by 12% of glycerol + egg yolk citrate extender (fraction B, Seshoka et al. 2012). Diluted semen samples were equilibrated for 4 h at 5°C. After the equilibration period, samples were loaded into 0.25-mL straws and transferred into a controlled rate programmable freezer. After the target temperature of –130°C was reached, semen straws were stored in a LN tank (–196°C). After 3 months of storage, straws were thawed at 15°C (first and second freezing and thawing followed the same process) for 5 min and further evaluated post-thawed at 0 and 15 min during incubation at 15°C. Treatment means were separated using Fisher’s protected t-test least. No significant differences were recorded between the raw semen total sperm motility percentage (93.2%) and first frozen-thawed at 0 min (82.6%), with the total sperm motility rate recovery of 88.6%. In addition, there was a marked decline recorded in sperm total motility during the first frozen-thawed at 15 min (77.6%), second frozen-thawed at 0 min (31.3%), and second frozen-thawed at 15 min (30.1%; P < 0.05). The sperm curvilinear velocity and average path velocity was reduced following first frozen-thawed (P < 0.05) but remained constant and stable between the treatment groups (P > 0.05). In conclusion, the freezing-thawing process did not reduce the Nguni bull total sperm motility during the first freezing and thawing process, compared with raw semen. However, a drastic decline was recorded during the second freezing-thawing processes, compared with raw semen.

76 THE EFFECT OF GLYCEROL CONCENTRATIONS IN EGG-YOLK CITRATE EXTENDER ON THE QUALITY OF CRYOPRESERVED NGUNI BULL SEMEN

2013 ◽  
Vol 25 (1) ◽  
pp. 185
Author(s):  
M. M. Seshoka ◽  
M. L. Mphaphathi ◽  
F. V. Ramukhithi ◽  
T. R. Netshirovha ◽  
C. Hlungwani ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Rural Areas ◽  
Egg Yolk ◽  
Positive Control ◽  
Negative Control ◽  
Glycerol Concentration ◽  
Bull Semen ◽  
Frozen Semen ◽  
Thawing Process ◽  
Motility Rate

There are bull shortages in South African poor rural areas. Artificial-insemination technology could play a significant role on breeding emerging farmer’s cattle. The objective of this study was to compare glycerol concentrations (0, 4, 8, or 12%) during freezing of Nguni bull semen to conduct AI in different villages. Semen was collected by electro ejaculator from 2 Nguni bulls of known and proven fertility. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories within 10 min after collections. Semen samples were pooled and evaluated by Sperm Class Analyser® and allocated randomly per treatment. Semen was then diluted (1 : 2 v:v) with egg-yolk citrate extender supplemented with either 0% (negative control), 4, 8, and 12% of glycerol concentration or AndroMed® (positive control). Semen samples were equilibrated for 4 h at 5°C. After equilibration period, samples were loaded into 0.5-mL straws and placed horizontally into the controlled rate (–5, –8, –10, –12, –15, –25, –35°C min–1) from 5°C until target temperature of –80°C is reached. The frozen semen straws were stored in a liquid nitrogen tank (–196°C) until thawing. Treatment means were separated using Fisher’s protected t-test least, and data are presented as mean ± SD. There was a significant differences (P < 0.05) between raw total sperm motility (83.3 ± 19.3) and frozen–thawed sperm with either 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), or 12% (61.5 ± 4.7) of glycerol and on AndroMed® (27.7 ± 17.8) group. Regardless of the glycerol concentrations used, the freezing-thawing process reduced (P < 0.05) the Nguni total sperm motility rate compared to uncryopreserved sperm (83.3 ± 19.3). In conclusion, egg-yolk citrate extender supplemented with 12% glycerol yielded a better (P < 0.05) total sperm motility rate (61.5 ± 4.7) as compared with the 0% (0.0 ± 0.0), 4% (30.2 ± 13.4), 8% (47.9 ± 12.5), and AndroMed® (27.7 ± 17.8) group. Further studies are required to test other levels of glycerol concentrations (>12%) on freezing Nguni semen and conducting AI.

53 COMPARISON OF 4 DIFFERENT CRYOPROTECTANTS ON FREEZING NGUNI BULL SPERMATOZOA EVALUATED BY COMPUTER-AIDED SPERM ANALYSIS

2014 ◽  
Vol 26 (1) ◽  
pp. 140
Author(s):  
M. M. Seshoka ◽  
M. L. Mphaphathi ◽  
T. L. Nedambale
Keyword(s):  
Sperm Motility ◽  
Artificial Insemination ◽  
Egg Yolk ◽  
Cleavage Rate ◽  
Sperm Analysis ◽  
Bull Semen ◽  
Significant Difference ◽  
Computer Aided ◽  
Freezing Curve ◽  
Motility Rate

Cryopreservation has been reported to damage approximately 40 to 50% of viable spermatozoa in bulls. It is critical to evaluate frozen-thawed spermatozoa with computer-aided sperm analysis (CASA) and find a suitable cryoprotectant for Nguni semen. The study was conducted to compare cryo-effectiveness of glycerol (GLY), dimethyl sulfoxide (DMSO), propanediol (PND), and ethylene glycol (EG) cryoprotectants at 12% concentrations during freezing of Nguni bull semen. Semen was collected from 18 stud Nguni bulls of proven fertility with the use of an electro-ejaculator. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories. Semen samples were pooled and sperm motility rate was evaluated using CASA. Semen was then diluted (1 : 2; vol : vol) with egg-yolk citrate extender supplemented with either 12% GLY, DMSO, PND, or EG. Semen samples were equilibrated for 4 h at 5°C. After equilibration, samples were loaded into 0.25-mL straws and placed into the controlled rate programmable freezer using a customized freezing curve (from 5 to –5°C at 0.008°C min–1 and from –3 to – 130°C at 6°C min–1). Following thawing of semen, artificial insemination was conducted on 104 oestrus-synchronised Nguni cows. The IVF was also conducted on 120 oocytes to check the cleavage rate. Data were analysed using ANOVA. There was a significant difference (P < 0.05) between raw total sperm motility (94.70 ± 2.63) and frozen-thawed sperm total motility with GLY (77.80 ± 11.03), EG (20.35 ± 11.86), DMSO (15.68 ± 10.14), and PND (11.19 ± 11.27) groups. The pregnancy rate following artificial insemination was 75.9% and a total of 86.6% oocytes had cleaved after fertilization with frozen (12% GLY)/thawed semen. In conclusion, cryopreservation process reduced sperm motility and velocity rates, regardless of cryoprotectant. Egg-yolk citrate extender supplemented with 12% glycerol had recorded the highest post-thaw sperm motility rate.

162 FREEZING OF SEMEN FROM ENDANGERED ASTURIANA DE LA MONTAÑA BULLS IN ZWITTERONIC LIPIDS-BASED EXTENDERS

2008 ◽  
Vol 20 (1) ◽  
pp. 161 ◽  
Author(s):  
C. Tamargo Miguel ◽  
S. S. Pérez-Garnelo ◽  
P. Beltrán Breña ◽  
A. T. Palasz ◽  
J. De la Fuente ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Semen Analysis ◽  
Egg Yolk ◽  
Semen Parameters ◽  
Computer Assisted ◽  
Progressive Motility ◽  
Bull Semen ◽  
Straight Line ◽  
Before And After ◽  
Arcsine Transformation

This experiment was designed to test the efficacy of 2 different preparation protocols of zwitteronic soyabean-origin lipids for the production of lipidsglycerol liposomes for use in bull semen cryopreservation. Lipids liposomes were prepared at 10% concentration in Tris buffer by 1. highpressure homogenization (Panda 2K, Parma, Italy) and then 8% glycerol were added, extender-1 (E-1); Lipids were homogenized together with glycerol, extender-2 (E-2). Bioxcell extender (E-3) was used as control. Semen was collected 3 times from 3 endangered Asturiana de la Monta�a bulls by the means of an artificial vagina. Ejaculates with at least 70% motility were processed further by a standard freezing protocol used in our AI station. Semen was diluted at 37�C with each of the 4 extenders to a concentration of 92 � 106 spermatozoa per mL, cooled to 4�C over 4 h, aspirated into 0.25-mL plastic straws (IMV Technologies, Aigle, France), frozen in a bio-freezer (IMV Technologies) in 3 steps from 4 to –140�C, and then plunged into liquid N2. Straws were thawed in a water bath at 37�C for 30 s. Sperm motility was analyzed microscopically immediately after collection, after dilution, and after 4, 24, 48, and 72 h of storage at 4�C. Post-thaw semen progressive motility was assessed microscopically, and sperm movement characteristics were analyzed by computer-assisted semen analysis (CASA) (SCA�, Microptic, Barcelona, Spain). Data were compared between extenders and bulls by 2-way ANOVA; percentages were transformed by arcsine transformation before ANOVA. Total and progressive sperm motility at 0 h after dilution ranged from 90 to 70% and was not different between extenders and bulls. There was no difference between bulls in total and progressive motility after 24 h of cold storage; however, both were significantly greater (P < 0.05) for Control (62.4 � 14.7 and 41.4 � 14.9) and E-1 (70.1 � 12 and 33.8 � 7.0) extenders than for the E-2 extender (22.5 � 17 and 1.2 � 1.3). Average post-thaw sperm motility was not different between bulls for either extender, but motility for Bioxcell (Control, 48.1 � 14.6%) and E-1 extenders (43.2 � 13.0%) were significantly greater (P < 0.05) than for E-2 extender (18.7 � 8.8%). There were no differences between bulls for all kinematic semen parameters: curvilinear (VCL), straight line (VSL), average path (VAP) velocities, linearity (LIN) and straightness (STR), evaluated by CASA before and after freezing; however, all were lower (P < 0.05) for the E-2 extender and not different between Control and E-1 extenders. Sperm movements derived from heads (VCL) and linearity of sperm(LIN), both closely related to field fertility, were in the range of 90.9 � 2.1 and 63.0 � 5.5 for E-3 (Control) extender; 99.1 � 3.4 and 49.4 � 3.5 for E-1; and 21.8 � 2.2 and 29.9 � 4.0 for E-2. In summary, zwitteronic soyabean lipid liposomes are an effective egg yolk substitute for the cryopreservation of Asturiana de la Monta�a bull semen; however, the homogenization protocol of the lipids-glycerol mixture must be improved.

38 QUAIL EGG YOLK IN CITRATE EXTENDER IS SUITABLE FOR CRYOPRESERVATION OF NGUNI BULL SEMEN

2016 ◽  
Vol 28 (2) ◽  
pp. 149
Author(s):  
M. M. Seshoka ◽  
M. L. Mphaphathi ◽  
K. S. Mafolo ◽  
M. Nkadimeng ◽  
Z. C. Raphalalani ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Egg Yolk ◽  
Sperm Analysis ◽  
Chicken Egg ◽  
Bull Semen ◽  
Significant Difference ◽  
Computer Aided ◽  
Chicken Egg Yolk ◽  
Fresh Semen ◽  
Motility Rate

Traditionally, commercial hen egg yolk has been used in extenders or freezing media because of its easy availability. However, the use of quail egg yolk has not been used for preserving Nguni bull semen. The aim of the study was to compare the suitability of different quail egg yolk concentrations (5, 10, 15, and 20%) for cryopreserving Nguni bull semen. Semen was collected from 14 stud Nguni bulls with the aid of electro ejaculator. Collected semen samples were kept in a thermos-flask containing warm water at a temperature of 37°C and transported to the laboratory for further analyses. The sperm motility traits were evaluated using computer-aided sperm analysis prior extension. Semen samples were then randomly allocated into 5, 10, 15, and 20% of quail egg yolk and 20% concentration of chicken egg yolk (control) in citrate extender. The extender was supplemented with 12% of glycerol (Seshoka et al. 2012) as a cryoprotectant, and semen samples were diluted (1 : 2) and equilibrated for a period of 4 h at 5°C. After equilibration, semen samples were loaded into 0.25-mL straws, placed into a controlled rate programmable freezer, and stored in a LN tank (–196°C) until thawing. Frozen semen straws were thawed in a water bath at 37°C for 1 min. Thawed semen was evaluated for sperm motility traits using a computer-aided sperm analysis system. Data were analysed with ANOVA. A significant difference was recorded between fresh total sperm motility rate (99%) and frozen-thawed semen samples with either 5% (87.3%) quail or 20% (87.6%) chicken egg yolk citrate extender compared with 10% (92.6%), 15% (91.2%), or 20% (89.9%) quail egg yolk citrate extender. Moreover, fresh semen also resulted in a significantly higher progressive sperm motility rate (39.3%) as compared with frozen-thawed with 5% (26.2%) or 20% quail (28.5%) or 20% chicken (22.7%) egg yolk citrate extender. The results also demonstrated that the use of 10, 15, and 20% quail egg yolk in citrate extender yielded comparable results on total sperm motility with fresh semen as compared with 5% quail and 20% chicken egg yolk. In conclusion, quail egg yolk extender provided sufficient cryo-effectiveness to cryopreservation of Nguni bull semen.

306 THE EFFECT OF BoviPURE GRADIENT ON BISON SPERM CRYOPRESERVATION

2010 ◽  
Vol 22 (1) ◽  
pp. 309
Author(s):  
O. A. Bogle ◽  
C. Lessard ◽  
R. B. McCorkell ◽  
T. Grafton ◽  
G. P. Adams
Keyword(s):  
Disease Status ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Gradient System ◽  
Separation And Purification ◽  
Freezing And Thawing ◽  
Sperm Analysis ◽  
Progressive Motility ◽  
Before And After ◽  
Wood Bison

Endemic tuberculosis and brucellosis threaten Canada’s wood bison population. For the purposes of developing a procedure to harvest pathogen-free sperm from bison, a sperm separation and purification product, BoviPure, was tested to determine its effect on bison sperm viability before and after cryopreservation. BoviPure (NidaCon International AB, Mölndal, Sweden) is a density centrifugation gradient system that contains trypsin and is designed to separate motile from non-motile sperm and remove infectious pathogens. Spermatozoa were expelled from the caudal epididymis of bison testicles collected at slaughter and diluted in 3 to 5 mL of TCM-199 medium (Gibco, Burlington, Ontario, Canada). After 1 h of incubation at 37°C, 1 to 1.5 mL of diluted sample (approximately 300 × 106 sperm) was placed in (1) a BoviPure gradient containing trypsin (n = 18 samples), (2) a BoviPure gradient without trypsin (n = 10 samples), or 3) left untreated (control; n = 20). Gradients were centrifuged at 300g for 20 min. The sperm pellet was resuspended in 5 mL of TCM-199 medium and recentrifuged at 500 g for 10 min. Total and progressive motility were estimated using computer-assisted sperm analysis (CASA). Gradient-treated and untreated sperm samples were diluted in an egg yolk extender (Triladyl; Minitube Canada, Ingersoll, Ontario, Canada), placed in 0.5-mL straws (50-200 × 106 spermatozoa per straw), frozen, and stored in liquid nitrogen for a minimum of 24 h. Semen straws were thawed by plunging in a 37°C water bath for 30 s, and motility measurements were taken immediately by CASA. Total and progressive motility were compared among groups by analysis of variance. Before cryopreservation, total motility and progressive motility were not affected by gradient processing. Total motility was 85±5.1%, 80±4.8%, and 77 ± 3.7% for untreated, BoviPure with trypsin, and BoviPure without trypsin, respectively (mean ± SEM), and progressive motility was 74 ± 2.7%, 77 ± 3.8%, and 68 ± 4.7%, respectively. After freezing and thawing, total motility decreased compared to unfrozen samples (P < 0.0001) but was not different among groups (37 ± 3.6%, 33 ± 3.7%, and 29 ± 4.8% for untreated, BoviPure with trypsin, and BoviPure without trypsin, respectively). Similarly, progressive motility decreased compared to unfrozen samples (P < 0.0001) but was not different among groups (26 ± 3.6%, 21 ± 3.7%, and 14.5 ± 4.9% for untreated, BoviPure with trypsin, and BoviPure without trypsin, respectively). We conclude that the BoviPure gradient with or without trypsin does not influence total motility or progressive motility of bison sperm either before or after cryopreservation and has potential as a method of harvesting pathogen-free sperm from wild animals of unknown disease status. This study was supported by Canadian Adaptation and Rural Development in Saskatchewan.

Effect of Addition of Moringa Oleifera Extract to Tris Extender on the Preservability of Cattle Bull Semen

2020 ◽  
Keyword(s):  
Free Radicals ◽  
Sperm Motility ◽  
Moringa Oleifera ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Natural Antioxidant ◽  
Herbal Supplement ◽  
Bull Semen ◽  
Before And After ◽  
Sperm Membrane

Moringa oleifera extract is a strong natural antioxidant that when was added to the semen extenders, it induced a cryoprotection to spermatozoa effect through elimination of the excess free radicals. So, the existing study intended for clarification of the consequence of extract of Moringa leaves (MLE) on bull spermatozoa after chilling and cryopreservation. MLE concentrations were 0% (control), 10, 20, 30, 40 and 50% (v/v) [MLE: TCF (Tris-citric-fructose diluent)] then 20% egg yolk was added, then the extended semen was assigned to the freezing protocol. Then, it was evaluated for (motility, alive, abnormality %, sperm membrane integrity % before and after freezing). Sperm motility was kept high with the concentration 10, 30 and 40% of MEEY till 8 days of chilling. The concentration 20% maintained sperm motility high till 7 days of chilling. Addition of MLE to TCF significantly (P<0.002) improved sperm motility in all concentrations except the 50% moringa enriched extender with egg yolk (MEEY) where sperm motility was maintained as the control. The use of MEEY maintained % of alive sperms and % of normal spermatozoal membrane (HOST%) as good as the control. In conclusion: moringa as a herbal supplement to semen diluents enhanced preservation in cooled and cryopreserved cattle bull semen.

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.

scholarly journals Determination of the optimal concentration of Minitube Equex paste for boar semen cryopreservation based on sperm motility characteristics

2020 ◽  
Vol 52 (3) ◽  
Author(s):  
Junpen Suwimonteerabutr ◽  
Morakot Nuntapaitoon ◽  
Padet Tummaruk
Keyword(s):  
Sperm Motility ◽  
Egg Yolk ◽  
Group Iv ◽  
Optimal Concentration ◽  
Freezing And Thawing ◽  
Semen Cryopreservation ◽  
Group Iii ◽  
Group I ◽  
Boar Semen ◽  
Group Ii

Equex paste is a non-permeating cryoprotective agent (CPA) that improved post-thaw survival of spermatozoa during boar semen cryopreservation. However, Equex paste produced by Nova Chemical Sales Inc. (MA, USA) is not currently available. The aim of the present study was to determine the optimal concentration of Minitube Equex paste (Minitube, Tiefenbach, Germany) for boar semen cryopreservation in comparison with Nova Equex STM paste (control). Fifteen ejaculates from 12 mature boars were collected by the glove-hand method. Each ejaculate was aliquoted and cryopreserved in base freezing extender III as Tris-citrate egg yolk (TEY) extender plus 9.0% glycerol classified into four groups. Group I was the control and included only 1.5% Nova Equex STM paste. Groups II, III and IV were the experiment groups, and contained different concentrations of Minitube Equex paste (Group II: 1.5%; Group III: 1.7%; and Group IV: 1.9%) added to the freezing extender III. After freezing and thawing, sperm motility characteristics were evaluated by Sperm Class Analyzer® incubated at 37 °C for 0 (10 min), 1 and 2 h post-thawing. In Group IV after thawing at 0 h, rapid velocity and the velocity curved line were significantly higher than in Groups II and III (P &lt; 0.05) but did not differ from Group I. Moreover, after thawing at 1 h, LIN (linearity) in Group IV was higher than in Group II (P &lt; 0.05), but did not differ from the other groups. In conclusion, the most suitable concentration of Minitube Equex paste in the current protocol was 1.9% supplemented with 9.0% glycerol in TEY-based freezing extender III, based on the conformity between data from manual guides and the observed sperm motility characteristics results.

58 EFFECT OF MELATONIN IMPLANT ON BLANCA de RASQUERA BUCKS DURING SPRING ON SPERM MORPHOMETRY BEFORE AND AFTER CRYOPRESERVATION

2015 ◽  
Vol 27 (1) ◽  
pp. 122
Author(s):  
A. Tabarez ◽  
W. García ◽  
M. J. Palomo
Keyword(s):  
Egg Yolk ◽  
Sperm Head ◽  
Head Width ◽  
Computer Assisted ◽  
Sperm Cryopreservation ◽  
Sperm Analysis ◽  
Know How ◽  
Before And After ◽  
Sperm Morphometry ◽  
Computer Assisted Sperm Analysis

In order to improve sperm cryopreservation throughout the year and accelerate the process of preservation of this Catalonian goat breed in extinction danger, we proposed to assess the effect of melatonin implant application in Blanca de Rasquera males during spring on sperm head morphometry of fresh and thawed sperm. Therefore 8 bucks of 30 months old approximately were divided into 2 groups. In one of the groups, 2 melatonin implants (Melovine®, CEVA) were inserted into bucks 60 days before starting the collection of semen, and the other group was kept untreated. Briefly, fresh ejaculates from each group of 4 bucks were collected in spring, immediately mixed in equal quantities, and centrifuged twice (600 g for 10 min). Then the pellet was resuspended in a Tris-based medium containing 15% (v/v) of powdered egg yolk supplemented with 5% glycerol. Afterward, sperm samples were refrigerated at 5°C for 4 h before being frozen in LN vapour. Buck sperm head morphometry was analysed by computer-assisted sperm analysis (ISAS®) on fresh and thawed sperm previously stained with Diff Quick®. Data were analysed by GLM multivariate procedure (IBM SPSS, 2011; mean ± s.e., n = 6), showing significant differences among treatments in all the morphometric parameters except head perimeter and rugosity (Table 1). Our results suggest that melatonin application in bucks increases the ellipticity and elongation of fresh and thawed sperm, meanwhile the cryopreservation process reduces both parameters. Likewise melatonin implants increase significantly the head length only on thawed sperms as cryopreservation process increases the head width, area in sperms from implanted males and regularity only in sperms from nonimplanted bucks. These head changes on fresh and thawed sperm morphometry should be deeply investigated in order to know how they could affect sperm cryosurvival and fertility. Table 1.Effect of melatonin implant on Blanca de Rasquera bucks during spring on morphometry of fresh and thawed sperm This research was supported by INIA (RZ2009–00008–00–00), Generalitat de Catalunya (2009SGR0621 and CUR-DIUE), and FSE and Fundación Carolina.

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