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

77 ADDITION OF CHOLESTEROL IN FRESH GOAT SPERM IMPROVES CRYOSURVIVAL RATES

2013 ◽  
Vol 25 (1) ◽  
pp. 186
Author(s):  
B. G. Silva ◽  
E. A. Moraes ◽  
C. S. Oliveira ◽  
W. D. Ferrari Junior ◽  
W. C. G. Matos ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Sperm Motility ◽  
Phase Contrast ◽  
Room Temperature ◽  
Petri Dish ◽  
Egg Yolk ◽  
Irreversible Damage ◽  
Hypoosmotic Swelling Test ◽  
Contrast Microscopy ◽  
Working Solution

Cryopreservation causes irreversible damage to goat sperm membranes, measured by a loss of motile and functional normal cells, compared with fresh sperm. The objective of this study was to determine if the addition of cholesterol-loaded cyclodextrin (CLC) to goat semen improved sperm cryosurvival. The CLC was prepared as described by Purdy and Graham (2004 Cryobiology 48, 36–45) with some modifications: 200 mg of cholesterol were dissolved in 1 mL of chloroform and 1 g of methyl-beta-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass Petri dish and the solvents allowed to evaporate on a warm plate for 24 h. The resulting crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL TALP at 37°C. Thirty ejaculates from 5 bucks were collected, diluted 1 : 1 in Tris diluent, divided into 7 equal aliquots, and centrifuged at 800g for 10 min. The sperm pellets were resuspended in Tris diluent, to which 0, 0.75, 1.5, 3.0, 4.5, 6.0, or 7.5 mg of CLC/120 million sperm were added. All treatments were incubated for 15 min at room temperature and then cooled to 4°C over 2 h. The samples were then diluted with Tris-egg-yolk diluent containing 2% glycerol, and the sperm were packaged into 0.5-mL straws, frozen in static liquid-nitrogen vapour for 20 min, and plunged into liquid nitrogen. Straws were thawed in 37°C water for 30 s, extended in Tris, and analyzed using optic microscopy. To test thermal resistance, after thawing, 0.5 mL of semen from each treatment were placed in 1.5-mL Eppendorf tubes in a water bath at 37°C for 3 h. At 0, 60, 120, and 180 min, subsamples were evaluated for sperm progressive motility. A hyposmotic test was also conducted by adding 10 µL of sperm to 2 mL of each solution and incubating them for 1 h/37°C. Sequentially, 20 µL of sperm was diluted in hypoosmotic solution (150 mOsm), and the samples were evaluated using phase-contrast microscopy. A total of 100 spermatozoa were counted in at least 5 different fields, and sperm tails were classified as either noncoiled or coiled. Data were analyzed using ANOVA, and treatment means were separated, using the SNK test at 5% probability. The sperm motility (50.4, 33.8, and 22.5%) was significantly higher for sperm treated with 0.75 mg of cholesterol after 0, 60, and 120 min of incubation after thawing, when compared with other treatments. No treatment differences in the hypoosmotic swelling test were observed. The addition of 0.75 mg of cholesterol to fresh goat semen improved sperm motility after cryopreservation for up to 3 h. Supported by FACEPE and CAPES.

71 EFFECT OF CHOLESTEROL-LOADED CYCLODEXTRINS ON PRE- AND POST-THAW FUNCTION OF FELID SPERM

2015 ◽  
Vol 27 (1) ◽  
pp. 128 ◽  
Author(s):  
I. A. Plourde ◽  
H. L. Bateman ◽  
W. F. Swanson
Keyword(s):  
Sperm Motility ◽  
Calcium Ionophore ◽  
Fluorescein Isothiocyanate ◽  
Cholesterol Content ◽  
Domestic Cat ◽  
Sperm Cryopreservation ◽  
Soy Lecithin ◽  
Acinonyx Jubatus ◽  
Cholesterol Levels

Propagation of genetically diverse felid populations would benefit from more effective assisted reproduction strategies, including enhanced methods for sperm cryopreservation. In felids, sperm cryopreservation has been improved by substituting soy-lecithin for egg yolk in cryomedium (Vick et al. 2012 Theriogenology 78, 2120–2128). In other species, such as elephants (Kiso et al. 2012 Reprod., Fert. Dev. 24, 1134–1142) and cattle (Purdy et al. 2004 Cryobiology 48, 36–45), the addition of cholesterol-loaded cyclodextrins (CLC) to sperm before freezing has been shown to produce superior cryopreservation results. In this study, our objectives were to (1) assess cholesterol content of cat sperm membranes and capacitation status following incubation with CLC; (2) evaluate post-thaw sperm motility, acrosome status, and fertility in vitro following CLC treatment and freezing in a soy-based cryomedium; and (3) conduct a preliminary assessment of cholesterol content in nondomestic cat sperm. Freshly collected domestic cat sperm (n = 2 males, 3–4 ejaculates/male) were incubated with CLC (0, 1.5, or 3.0 mg mL–1), and cholesterol levels were measured using an Amplex Red Cholesterol Assay. Sperm aliquots from each CLC concentration were treated with calcium ionophore (2 μM, 30 min) during in vitro incubation and stained with fluorescein isothiocyanate/PNA to evaluate induced acrosomal loss. To assess post-thaw parameters, cat sperm treated with CLC were frozen in straws using soy-lecithin cryomedium, thawed, and cultured in vitro over time. To evaluate fertility, oocytes were collected laparoscopically from gonadotropin-treated domestic cats (n = 7 females, 147 oocytes total) and inseminated with low numbers of thawed-frozen sperm pretreated with 0 or 1.5 mg mL–1 CLC. Data were analysed using ANOVA and mean differences assessed with Fisher l.s.d. or chi-squared analysis. Sperm cholesterol levels were increased (P < 0.05) after exposure to both 1.5 and 3.0 mg mL–1 CLC. Prefreeze motility was decreased (P < 0.05) and capacitation was delayed at 3.0 mg mL–1 CLC relative to treatment with 0 or 1.5 mg mL–1 CLC. Both post-thaw motility and percentage of acrosome intact sperm were reduced (P < 0.05) with the highest CLC concentration, but results were similar (P > 0.05) for 0 and 1.5 mg mL–1 CLC. Fertilization percentages did not differ (P > 0.05) between treatment groups (0 CLC, 33.3%, 25/75; 1.5 mg mL–1 CLC, 26.4%, 19/72). Preliminary results from a single cheetah (Acinonyx jubatus) and single fishing cat (Prionailurus viverrinus) suggest that sperm membrane cholesterol may be lower compared to the domestic cat. Cholesterol content appeared to increase in both species after exposure to 1.5 mg mL–1 CLC. In summary, our findings suggest CLC treatment increased cholesterol content of felid sperm membranes. The higher CLC concentration was detrimental to sperm motility, capacitation, and post-thaw sperm traits. The lower CLC concentration did not improve post-thaw sperm function in domestic cats.Research supported by the Procter & Gamble Wildlife Conservation Scholarship Program.

164 CARNITINE IMPROVES POST-THAWING SPERM MOTILITY BY INCREASING ADENOSINE TRIPHOSPHATE CONTENT IN BUFFALO (Bubalus bubalis)

2017 ◽  
Vol 29 (1) ◽  
pp. 190
Author(s):  
V. Longobardi ◽  
G. Zullo ◽  
G. Albero ◽  
C. De Canditiis ◽  
A. Salzano ◽  
...  
Keyword(s):  
Adenosine Triphosphate ◽  
Sperm Motility ◽  
Critical Role ◽  
Automated System ◽  
Dna Integrity ◽  
Ammonium Compound ◽  
Dependent Manner ◽  
Atp Content ◽  
Freezing And Thawing ◽  
Semen Extender

Semen cryopreservation plays a critical role for a wide application of both AI and in vitro embryo production in buffalo. In this species, spermatozoa are more susceptible to hazards during freezing and thawing than cattle spermatozoa, thus resulting in lower fertilizing potential (Andrabi et al. 2008 Anim. Reprod. Sci. 104, 427–433). Carnitine is a quaternary ammonium compound with antioxidant capacities, able to reduce the availability of lipids for peroxidation by transporting fatty acids into the mitochondria for β-oxidation to generate adenosine triphosphate (ATP) energy (Tanphaichitr and Leelahagul 1993 Nutrition 9, 246–54). It is known that cryopreservation processes decreases the intracellular concentration of carnitine in spermatozoa (Reyes-Moreno et al. 2000 J. Androl. 21, 876–86). In cattle, supplementation of semen extender with carnitine improves sperm motility and DNA integrity (Bucak et al. 2010 Cryobiology 61, 248–53). The aim of this study was to evaluate whether supplementation of semen extender with carnitine would increase ATP content in buffalo sperm and affect post-thawing motility. Eight ejaculates from 4 bulls were used for the trial. Each ejaculate was split into 3 equal aliquots and diluted at 37°C with BioXcell extender containing 0 (control), 2.5, and 7.5 mM carnitine to a final concentration of 30 × 106 spermatozoa/mL. After 4 h at 4°C, the straws were frozen in an automated system. At thawing, sperm motility was evaluated by phase contrast microscopy at 40× magnification (Gillan et al. 2008 Anim. Reprod. Sci. 103, 201–204). Adenosine triphosphate content was measured using a Colourimetric ATP Assay Kit (Biovision, Milpitas, CA, USA). Briefly, Percoll-separated spermatozoa were homogenised and then deproteinized using 10-kDa spin columns. Samples were incubated at RT for 30 min and absorbance was measured at 570 nM in a microplate reader. Differences in sperm motility and ATP content among groups were analysed by ANOVA. Both concentrations of carnitine increased post-thawing sperm motility compared with the control (44.4 ± 3.5, 53.1 ± 3.9, and 52.5 ± 3.6, respectively, with 0, 2.5, and 7.5 mM carnitine; P < 0.05). Interestingly, carnitine increased ATP content of buffalo frozen–thawed sperm in a dose-dependent manner (4.1 ± 0.1, 5.3 ± 0.1, and 8.2 ± 0.4 nM × 108 sperm, respectively, with 0, 2.5, and 7.5 mM carnitine; P < 0.01). In conclusion, the enrichment of semen extender with carnitine improved post-thawing motility of buffalo sperm by boosting mitochondrial ATP production, hence providing energy for use by spermatozoa.

102 Sperm cryopreservation with a soy lecithin-based medium in black-footed cats (Felis nigripes) and sand cats (Felis margarita)

2019 ◽  
Vol 31 (1) ◽  
pp. 177
Author(s):  
L. M. Vansandt ◽  
A. Moresco ◽  
R. González ◽  
A. Miller ◽  
J. Newsom ◽  
...  
Keyword(s):  
Egg Yolk ◽  
Animal Protein ◽  
Domestic Cat ◽  
Significant Advance ◽  
Sperm Cryopreservation ◽  
Soy Lecithin ◽  
Sand Cat ◽  
Embryo Cleavage ◽  
Blastomere Number ◽  
Time Point

Felid semen has historically been frozen using an egg yolk-based cryopreservation medium (TEY). However, the use of egg introduces several potential concerns, such as variability in composition, microbial contamination, and regulatory issues. Our recent research has focused on developing an animal protein-free medium containing soy lecithin (SOY). Our studies revealed that SOY was superior to TEY for freezing domestic cat sperm and provided similar results for freezing ocelot, Pallas’ cat, and fishing cat sperm. The objective of this study was to compare SOY to the standard TEY for sperm cryopreservation in 2 wild cat species: the black-footed cat and sand cat. Semen was collected from adult male cats (n=6/species) via electroejaculation, split into 2 aliquots, centrifuged, resuspended in either SOY or TEY, slow-cooled, and frozen in straws over nitrogen vapor. Sperm motility [percent progressively motile (PPM); rate of progressive motility on 0-5 scale (RPM)] was evaluated at 0, 1, 3, 6, and 24h post-thaw and acrosome status (AC) was assessed at 0 and 6h post-thaw. Heterologous IVF was performed using oocytes collected laparoscopically from gonadotropin-treated domestic cats. At 48h post-insemination, Hoechst33342 staining was used to determine oocyte stage, number of blastomeres, and number of accessory sperm (AS) bound to the zona pellucida of embryos and mature oocytes. Percent progressively motile, RPM, and AC were analysed with repeated-measures ANOVA; embryo cleavage, blastomere number, and AS number were analysed with one-way ANOVA. All data are reported as least squares means±average standard error. In the black-footed cat, PPM, RPM, and AC of SOY-treated sperm (32.5±4.0% motile, 2.8±0.2 RPM, 41.8±4.1% intact; 0h) did not differ from TEY-treated sperm (44.2±4.0% motile, 2.8±0.2 RPM, 46.8±4.1% intact; 0h) at any post-thaw time point (P &gt; 0.05). Similarly, in the sand cat, post-thaw PPM, RPM, and AC of SOY-treated sperm (36.7±5.2% motile, 2.6±0.2 progression, 53.3±5.8% intact; 0h) did not differ from TEY-treated sperm (45.8±5.2% motile, 2.8±0.2 RPM, 51.0±5.8% intact; 0h) at any time point (P &gt; 0.05). In black-footed cats, neither embryo cleavage (34.1±10.9% SOY; 58.5±10.9% TEY), blastomere number (7.8±0.8 SOY; 6.3±0.8 TEY), nor AS (3.5±0.8 SOY; 1.7±0.8 TEY) differed between treatments (P &gt; 0.05). Sand cat results were similar, with no difference between SOY and TEY for cleavage (44.7±10.8% SOY; 40.6±10.8% TEY) or blastomere number (7.4±2.0 SOY; 6.7±2.0 TEY) (P &gt; 0.05), but AS was higher in SOY-treated sperm (4.3±0.2 SOY; 3.5±0.2 TEY, P=0.0183). These data collectively demonstrate that our SOY medium was an effective substitute to TEY for sperm cryopreservation in the black-footed cat and sand cat. The replacement of an egg yolk-based cryomedium with a chemically defined, animal protein-free alternative represents a significant advance in quality control and biosecurity for felid semen banking and should augment the use of assisted reproduction for population management of imperiled cats. Funded by the Institute of Museum and Library Services.

scholarly journals Comparison of Extenders With the Addition of Egg Yolk for Cooling Alpaca Sperm Obtained From Deferent Ducts

2020 ◽  
Vol 7 ◽  
Author(s):  
Mariana Lucía Bertuzzi ◽  
Edita Yola Torres ◽  
Teodosio Huanca ◽  
Deborah Neild ◽  
María Ignacia Carretero
Keyword(s):  
Sperm Motility ◽  
Chromatin Condensation ◽  
Egg Yolk ◽  
Final Concentration ◽  
Sperm Parameters ◽  
Membrane Function ◽  
Progressive Motility ◽  
Evaluation Time ◽  
Viable Sperm ◽  
Acrosome Integrity

The use of non-commercial and commercial extenders for cooling alpaca sperm has already been reported, the latter showing certain advantages over the first. The Andromed® (AM) extender was created for use in ruminants and has also been tested in ejaculated and epididymal alpaca sperm. According to the manufacturer, this extender does not need the addition of egg yolk (EY); however, it is known that the addition of EY to some extenders improves the preservation of cooled sperm. The objective of this study therefore was to compare a non-commercial extender (Tris) with the addition of EY vs. the commercial extender AM with and without the addition of EY, for cooling alpaca sperm obtained from diverted deferent ducts. Fifteen pools of deferent duct sperm were formed using samples from two or three different males for each. Each sperm pool was evaluated and then divided into three aliquots that were diluted to a final concentration of 30 × 106 sperm ml-1 (0 h) with either: (1) Tris with 20% EY (T-EY), (2) AM, or (3) AM with 20% EY (AM-EY). Samples were cooled to 5°C and the following sperm parameters were evaluated after 24 and 48 h of storage: motility, viability, membrane function, acrosome integrity, morphology, and chromatin condensation. Motility was also evaluated after 72 h of storage. The samples that best preserved progressive and total sperm motility at the 24 and 48 h evaluation periods were the ones diluted with AM-EY, observing that with this extender these motility patterns decreased significantly after 72 h of storage compared to time 0 h (p &lt; 0.05). A significant decrease (p &lt; 0.05) in total and progressive motility was observed at 48 h for the T-EY and AM extender compared to 0 h. AM was the only extender in which the percentages of viable sperm decreased significantly (p &lt; 0.05) after 48 h of conservation. For the rest of sperm parameters evaluated, no significant differences were observed between any of the extenders at any evaluation time. The Andromed® extender with the addition of 20% EY could be an alternative option for cooling alpaca sperm obtained from deferent ducts.

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.

scholarly journals Efficiency of Commercial Egg Yolk-Free and Egg Yolk-Supplemented Tris-Based Extenders for Dromedary Camel Semen Cryopreservation

Animals ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 999 ◽  
Author(s):  
Ayman Abdel-Aziz Swelum ◽  
Islam M. Saadeldin ◽  
Hani Ba-Awadh ◽  
Mohsen G. Al-Mutary ◽  
Abdullah F. Moumen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Dromedary Camel ◽  
Plasma Membrane Integrity ◽  
Semen Cryopreservation

This study compared the efficiency of commercial egg yolk-free (AndroMed, OPTIXcell) and egg yolk-supplemented (Triladyl, Steridyl) Tris-based extenders for semen cryopreservation in seven adult dromedary camels. The camel-specific extender SHOTOR was used as control. The collected semen samples were evaluated and diluted with SHOTOR, Triladyl, Steridyl, AndroMed, or OPTIXcell. The diluted semen was gradually cooled and equilibrated for two hours before liquid nitrogen freezing. Semen was evaluated prior to freezing and after freeze-thawing cycles for motility, kinetics, vitality, abnormality, plasma membrane integrity, and DNA fragmentation using computer-assisted sperm analysis. In pre-freezing evaluation, progressive sperm motility was higher in SHOTOR-diluted samples (21.54 ± 1.83) than in samples diluted with Steridyl, OPTIXcell, or AndroMed (15.76 ± 1.80, 17.43 ± 1.10, and 13.27 ± 1.07, respectively). Moreover, Triladyl and SHOTOR resulted in significantly (p < 0.05) better sperm vitality and DNA integrity than all other diluents, but Triladyl resulted in a significantly (p < 0.05) better plasma membrane integrity (87.77 ± 0.31) than SHOTOR (85.48 ± 0.58). In the post-thawing evaluation, Triladyl led to significantly (p < 0.05) higher sperm motility (38.63 ± 0.81%; p < 0.05) when compared to SHOTOR, Steridyl or AndroMed (35.09 ± 1.341%, 34.4 ± 0.84%, and 31.99 ± 1.48%, respectively), with OPTIXcell being the least efficient (28.39 ± 0.86%). Progressive sperm motility was the highest when using Triladyl. Post-thawing curvilinear, straight line and average path sperm velocities were highest with Triladyl and lowest with AndroMed. Triladyl led to the highest linearity coefficient and straightness sperm coefficient, while SHOTOR to the highest DNA and plasma membrane integrity. OPTIXcell and AndroMed resulted in poor post-thawing sperm vitality, while Steridyl was less efficient than Triladyl. The highest rate of sperm abnormalities was recorded with OPTIXcell and the lowest with SHOTOR or Triladyl. In conclusion, SHOTOR, Triladyl, Steridyl, AndroMed, and OPTIXcell can all be used for camel semen cryopreservation; however, SHOTOR and Triladyl provided the best post-thawing sperm quality. Based on our findings, Triladyl is the best commercially available extender for dromedary camel semen cryopreservation to date.

21 Effect of egg yolk extracted low-density lipoprotein on cryopreserved Nguni bull semen

2019 ◽  
Vol 31 (1) ◽  
pp. 136
Author(s):  
M. M. Tshabalala ◽  
K. A. Nephawe ◽  
M. L. Mphaphathi ◽  
C. M. Pilane ◽  
T. L. Nedambale
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Semen Parameters ◽  
Dna Integrity ◽  
Low Density ◽  
Bull Semen ◽  
Frozen Semen ◽  
Live Sperm

Egg yolk has been reported to have a beneficial effect on sperm quality following cryopreservation, and this led to its widespread use in semen extenders. However, egg yolk contains substances that inhibit respiration of sperm cells and diminish their motility rate. Moreover, it also contains low-density lipoproteins (LDL) that have a protective effect on sperm during the cryopreservation process. The objective of this study was improve cryopreservation of Nguni bull semen using egg yolk low-density protein. A total of 25 ejaculates were collected from 5 Nguni bulls aged 4 to 5 years using an electroejaculator during the natural breeding season. Collected raw semen samples were transported to the laboratory and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity before dilution. Semen was randomly diluted with a sodium citrate extender supplemented with 20% egg yolk (control) and with 6, 8, 10, and 12% LDL concentrations. The diluted semen sample groups were equilibrated for 4h at 5°C. Following equilibration, semen was loaded into 0.25-mL straws and frozen in a controlled-rate programmable freezer. The groups of semen straws were then plunged into LN and transferred into LN tanks (−196°C) for storage. The frozen semen straws per treatment group were thawed at 37°C and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity. Data were analysed with ANOVA using Stata V12 statistical software (StataCorp., College Station, TX), and treatment means were separated using Fisher’s protected t-test at the significant level of P&lt;0.05. Sperm motility and membrane integrity were significantly higher (P&lt;0.05) in frozen-thawed semen diluted with 8% LDL compared with the other concentrations. However, 6 and 8% LDL resulted in a significantly higher (P&lt;0.05) live sperm, DNA, and acrosome integrity. Frozen-thawed semen diluted with 10 and 12% LDL resulted in the lowest percentages of sperm motility, live sperm, plasma membrane, acrosome, and DNA integrity following cryopreservation. In conclusion, extender containing 8% LDL resulted in improved Nguni bull semen parameters such as sperm motility, viability, plasma membrane, acrosome, and DNA integrity following cryopreservation. Further studies are required to determine the fertilizing capacity of semen diluted and frozen with LDL in vitro and in vivo.

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.

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