scholarly journals Optimization of Sperm Cryopreservation Protocol for Peregrine Falcon (Falco peregrinus)

Animals ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 691
Author(s):  
Beatriz Cardoso ◽  
Irene Sánchez-Ajofrín ◽  
Cristina Castaño ◽  
Olga García-Álvarez ◽  
Milagros Cristina Esteso ◽  
...  
Keyword(s):  
Mitochondrial Activity ◽  
Peregrine Falcon ◽  
Sperm Cryopreservation ◽  
Freezing Method ◽  
Ultrarapid Freezing ◽  
Complex Process ◽  
Acrosome Integrity ◽  
Cryopreservation Protocol ◽  
Motility Parameters ◽  
Important Progress

Sperm cryopreservation is a complex process that needs to be adapted to wild and domestic avian species to ensure proper efficiency. Because of its accessibility, the peregrine falcon may be used as a good model for studying other raptor species. To find the most optimal cryopreservation protocol for peregrine falcon ejaculates, sperm parameters such as motility, viability, DNA fragmentation, acrosome integrity, and mitochondrial activity were analyzed under different conditions by varying the freezing method (slow freezing in straws vs. ultrarapid freezing in pellets), thawing conditions (37 °C for 30 s vs. 5 °C for 1 min), type of cryoprotectant (DMA vs. DMSO), and concentration of DMSO (4% vs. 8%). Results show that slow cryopreservation in straws yielded greater percentages (p < 0.05) of motile spermatozoa (22.5% ± 4.4% vs. 0.0% ± 4.1%), viable spermatozoa with intact acrosomes (84.6% ± 4.3% vs. 77.4% ± 4.3%), and spermatozoa with active mitochondria (41.0% ± 6.7% vs.12.8% ± 6.7%), compared with those obtained by the ultrarapid freezing in pellets. However, no differences were found between different thawing conditions. Moreover, all sperm motility parameters were greater (p < 0.05) when DMSO was used during freezing compared with DMA, although the use of 3% and 8% DMSO produced similar results. In conclusion, these results represent important progress in the study of falcon semen cryopreservation protocol, highlighting the crucial steps of the process and the most suitable conditions.

92 Sperm Cryopreservation in the Burmese Python (Python bivittatus) as a Model for Endangered Snakes

2018 ◽  
Vol 30 (1) ◽  
pp. 185
Author(s):  
C. Young ◽  
N. Ravida ◽  
M. Rochford ◽  
B. Durrant
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Monitoring Program ◽  
Florida Everglades ◽  
Equal Weight ◽  
Sperm Cryopreservation ◽  
Acrosome Integrity ◽  
Python Bivittatus ◽  
Southern Florida ◽  
Cryopreservation Protocol

The Burmese python (Python bivittatus) is listed as vulnerable by the International Union for Conservation of Nature (IUCN). Released pet Burmese pythons have detrimental effects on fauna native to southern Florida and are responsible for localised declines of several species in some parts of the Everglades National Park (IUCN, 2012; 10.2305/IUCN.UK.2012-1.RLTS.T193451A2237271.en). As part of an invasive species monitoring program, Burmese pythons were captured in the Florida Everglades and used as a model for the development of sperm cryopreservation protocols for endangered snakes. Sperm was collected by flushing the vas deferens postmortem and initial motility score (IMS; % motile × speed of progression2), plasma membrane integrity (IPL), and acrosome integrity (IAC) were recorded before cryopreservation. Sperm was extended in TEST-yolk buffer with final dimethyl sulfoxide (DMSO) or glycerol (GLY) concentrations of 8, 12, or 16%, or combinations of DMSO and GLY with final concentrations of 4:4, 6:6, or 8:8%. Sperm in 500 µL of extender was frozen in vials at 0.3°C/min to –40°C before storage in liquid nitrogen. For each treatment, triplicate vials from each of 3 males were thawed at 37°C for 90 s. Cryoprotectant was removed by centrifugation and the sperm pellet was resuspended in TCM-199+HEPES. Sperm was evaluated at 22°C immediately following resuspension (T0) and at 60 (T60) minutes. All data were expressed as a percentage of initial (%IMS, %IPL, and %IAC). The effects of freeze method on %IMS, %IPL and %IAC were analysed by ANOVA and Tukey’s HSD test. Freeze method significantly affected %IMS at T0 (P = 0.0004) and T60 (P = 0.0001), with sperm frozen in the 6%DMSO:6%GLY and 4%DMSO:4%GLY treatments resulting in the highest %IMS at both T0 (19.4% and 17.7%, respectively) and T60 (26.7% and 14.4%, respectively). Regardless of cryoprotectant concentrations, sperm frozen in a combination of DMSO and GLY exhibited significantly higher %IMS than all treatments of DMSO or GLY alone (P < 0.0001 at T0 and T60). The %IPL was significantly affected by freeze method at T0 (P < 0.0001) and T60 (P = 0.0266). Sperm frozen in 8%DMSO:8%GLY and 6%DMSO:6%GLY retained greater %IPL at both T0 (69.1% and 65.7%, respectively) and T60 (47.8% and 49.9%, respectively). Acrosome integrity was significantly affected by freeze method at T0 (P < 0.0001) and sperm frozen in 8% DMSO resulted in the greatest %IAC (56.4%). In addition, all DMSO and DMSO:GLY treatments preserved a significantly greater proportion of intact acrosomes than GLY alone (P < 0.0001). To simplify these analyses and to determine the best overall freeze method for this species, a sperm quality index (SQI) was calculated, giving equal weight to each of the 3 measured indicators of cryosurvival. The SQI analysis revealed that Burmese python sperm frozen at 0.3°C/min in either 6%DMSO:6%GLY or 4%DMSO:4%GLY exhibited significantly higher post-thaw viability at T0 and T60 than all other treatments. This study represents the first comparative, comprehensive attempt to develop a sperm cryopreservation protocol for any snake species.

scholarly journals Effects of Cryoprotective Medium Composition, Dilution Ratio, and Freezing Rates on Spotted Halibut (Verasper variegatus) Sperm Cryopreservation

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 2153
Author(s):  
Irfan Zidni ◽  
Yun Ho Lee ◽  
Jung Yeol Park ◽  
Hyo Bin Lee ◽  
Jun Wook Hur ◽  
...  
Keyword(s):  
Previous Experiment ◽  
Activity Index ◽  
Medium Composition ◽  
Freezing Rate ◽  
Dilution Ratio ◽  
Sperm Cryopreservation ◽  
Ringer’S Solution ◽  
Potential Market ◽  
Spotted Halibut ◽  
Cryopreservation Protocol

The spotted halibut is species that has a high potential market value in Korea, but the supply of seed is unstable because of the limited milt production of males. The objective of this research was to explore different aspects, such as CPAs, diluents, dilution ratio, and freezing rates, to develop an optimal sperm cryopreservation. The parameters assessed were movable sperm ratio, sperm activity index, survival rate, and DNA damage. The CPAs tested in this research were propylene glycol, dimethyl sulfoxide (DMSO), methanol, ethylene glycol, and glycerol. Different diluents, including 300 mM sucrose, 300 mM glucose, Stain’s solution, and Ringer’s solution, were investigated. The previous experiment showed that the optimal CPA for cryopreservation was DMSO with a concentration of 15% with 300 mM as diluent. To determine the effect of the dilution ratio, sperm was diluted to 1:1, 1:2, 1:10, 1:100, and 1:1000 with 300 mM sucrose containing DMSO at a final concentration of 15%. Lastly, the optimal freezing rate of the sperm was evaluated with four different freezing rates (−1, −5, −10, and −20 °C/min). Post-thaw sperm motility was higher with a dilution ratio lower than 1:2, and the freezing rate was less than −5 °C/min. In conclusion, these findings represent the development of a cryopreservation protocol for spotted halibut.

scholarly journals Acrosomal integrity and capacitation are not influenced by sperm cryopreservation in the giant panda

Reproduction ◽  
2004 ◽  
Vol 127 (5) ◽  
pp. 547-556 ◽  
Author(s):  
R E Spindler ◽  
Y Huang ◽  
J G Howard ◽  
P Wang ◽  
H Zhang ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Giant Panda ◽  
Calcium Ionophore ◽  
Sperm Cryopreservation ◽  
Management Tools ◽  
Giant Pandas ◽  
Before And After ◽  
Cryopreservation Protocol ◽  
Acrosomal Integrity

Sperm cryopreservation and artificial insemination are important management tools for giant panda breeding and the preservation of extant genetic diversity. This study examined the influence of freeze–thawing on sperm function, specifically capacitation. Sperm from nine giant pandas were assessed before and after rapid (− 40 and − 100 °C/min) cryopreservation by incubation in HEPES-buffered Ham’s F10 medium with and without the capacitation accelerators, 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP). At 0, 3 and 6 h of exposure, aliquots were assessed for sperm motility traits and capacitation, defined as the proportion of sperm with intact acrosomes following exposure to solubilised zonae pellucidae (ursid or felid) or calcium ionophore subtracted from the proportion of sperm with intact acrosomes before exposure. Although mean±s.e.m. sperm motility post-thaw (56.1 ± 3.9% at 0 h) was less (P < 0.05) than pre-freeze (71.7 ± 6.0%), there was no difference (P > 0.05) in the proportion of acrosome-intact sperm (fresh, 93.0 ± 1.7% versus cryopreserved–thawed, 81.7 ± 4.7% at 0 h). Incidence of capacitation was greater (P < 0.05) in fresh sperm incubated with capacitation accelerators IBMX and dbcAMP (9 h: 50.9 ± 1.1) compared with fresh sperm incubated without accelerators (9 h: 41.2 ± 1.1%). Frozen–thawed sperm preincubated without accelerators underwent capacitation (49.6 ± 1.1%) to a greater extent (P < 0.05) compared with these fresh counterparts. Thawed samples with (9 h: 45.9 ± 1.4%) and without accelerators (9 h: 41.2 ± 1.1%) did not differ (P > 0.05) during the 9-h incubation. We conclude that giant panda spermatozoa (1) undergo capacitation in vitro with or without chemical accelerators and (2) withstand a rapid cryopreservation protocol, including retaining normal acrosomal integrity and functional capacitation ability.

Development of a sperm cryopreservation protocol for the Argentine black and white tegu (Tupinambis merianae)

2017 ◽  
Vol 87 ◽  
pp. 55-63 ◽  
Author(s):  
Carly Young ◽  
Nicole Ravida ◽  
Michelle Curtis ◽  
Frank Mazzotti ◽  
Barbara Durrant
Keyword(s):  
Sperm Cryopreservation ◽  
Black And White ◽  
Tupinambis Merianae ◽  
Cryopreservation Protocol

Dimethylacetamide can be used as an alternative to glycerol for the successful cryopreservation of koala (Phascolarctos cinereus) spermatozoa

2008 ◽  
Vol 20 (6) ◽  
pp. 724 ◽  
Author(s):  
Yeng Peng Zee ◽  
William V. Holt ◽  
Jaime Gosalvez ◽  
Camryn D. Allen ◽  
Vere Nicolson ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Membrane Integrity ◽  
Sperm Nucleus ◽  
Sperm Chromatin ◽  
Similar Degree ◽  
Sperm Cryopreservation ◽  
Phascolarctos Cinereus ◽  
Degree Of Protection ◽  
Fish Sperm ◽  
Cryopreservation Protocol

Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of intermolecular disulfide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulfide bonds within their chromatin, but have been successfully cryopreserved. The present study examined the hypothesis that the cryoprotectants used for fish sperm cryopreservation would confer a similar degree of protection on koala spermatozoa. Three concentrations each of five cryoprotectants (dimethyl sulfoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide (DMA)) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Post-thaw assessment of progressive motility, plasma membrane integrity and mitochondrial membrane potential (MMP) revealed that protocols using 15% DMA achieved 62.2 ± 3.6% (P < 0.05) sperm survival, of which 79% (P < 0.05) had high MMP, an improvement of 32% and 40%, respectively, over sperm frozen in 14% glycerol. The percentage of spermatozoa with swollen nuclei was also lowest when frozen in 15% DMA, both immediately after thawing (18.0 ± 3.5%; P < 0.05) and after 2 h incubation at 35°C (35.8 ± 4.4%; P < 0.05). A second study was conducted to determine the optimal concentration of DMA for use in the cryopreservation of koala spermatozoa. High DMA concentrations (17.5% and 20%) resulted in significantly lower proportions of live spermatozoa showing high MMP immediately after thawing compared with spermatozoa frozen in the lower concentrations. The percentage of koala spermatozoa with swollen chromatin following cryopreservation was not affected by DMA concentration.

Immunolocalisation and expression of oxytocin receptors and sex hormone-binding globulin in the testis and epididymis of dogs: correlation with sperm function

2019 ◽  
Vol 31 (9) ◽  
pp. 1434
Author(s):  
Andressa Dalmazzo ◽  
João D. A. Losano ◽  
Daniel S. R. Angrimani ◽  
Isabel V. A. Pereira ◽  
Marcelo D. Goissis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Expression ◽  
Membrane Integrity ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Mitochondrial Activity ◽  
Hormone Binding ◽  
Plasma Membrane Integrity ◽  
Gene And Protein Expression ◽  
Acrosome Integrity

The aim of this study was to confirm gene and protein expression of oxytocin receptor (OTR) and sex hormone-binding globulin (SHBG) in the testis and epididymis of dogs, correlating these data with sperm quality and production and testosterone concentrations. Positive correlations were found between OTR and SHBG expression in both the testis and epididymis. Testicular OTR expression was positively associated with plasma membrane and acrosome integrity in canine spermatozoa, whereas SHBG expression in the testis was positively correlated with various sperm characteristics, such as sperm concentration, total and progressive motility, plasma membrane integrity and acrosome integrity. Testicular expression of both OTR and SHBG was negatively correlated with low sperm mitochondrial activity. In the epididymis, SHBG expression was only positively correlated with plasma membrane integrity. Analysis of protein expression revealed that testicular OTR was positively correlated with testosterone concentrations and negatively correlated with the absence of sperm mitochondrial activity. In addition, SHBG expression in the testes was associated with epididymis SHBG expression and morphologically normal cells. Immunohistochemical (IHC) analysis revealed the presence of both OTR and SHBG in testicular smooth muscles and Leydig cells. However, in the epididymis, OTR was only located in smooth muscle cells, whereas neither IHC nor western blotting detected SHBG. Together, the results of this study suggest that OTR and SHBG play key roles in spermatogenesis and sperm maturation, being essential for male reproductive success.

scholarly journals Conditioned Medium from Canine Amniotic Membrane-Derived Mesenchymal Stem Cells Improved Dog Sperm Post-Thaw Quality-Related Parameters

Animals ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1899
Author(s):  
Feriel Yasmine Mahiddine ◽  
Jin Wook Kim ◽  
Ahmad Yar Qamar ◽  
Jeong Chan Ra ◽  
Soo Hyun Kim ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Conditioned Medium ◽  
Amniotic Membrane ◽  
Membrane Integrity ◽  
Mitochondrial Activity ◽  
Flow Cytometry Analysis ◽  
Sperm Cryopreservation ◽  
Control Groups ◽  
Freezing Medium ◽  
And Control

This study investigated the effects of conditioned medium (CM) from canine amniotic membrane-derived MSCs (cAMSCs) on dog sperm cryopreservation. For this purpose, flow cytometry analysis was performed to characterize cAMSCs. The CM prepared from cAMSCs was subjected to proteomic analysis for the identification of proteins present in the medium. Sperm samples were treated with freezing medium supplemented with 0%, 5%, 10%, and 15% of the CM, and kinetic parameters were evaluated after 4–6 h of chilling at 4 °C to select the best concentration before proceeding to cryopreservation. Quality-related parameters of frozen–thawed sperm were investigated, including motility; kinetic parameters; viability; integrity of the plasma membrane, chromatin, and acrosome; and mitochondrial activity. The results showed that 10% of the CM significantly enhanced motility, viability, mitochondrial activity, and membrane integrity (p < 0.05); however, the analysis of chromatin and acrosome integrity showed no significant differences between the treatment and control groups. Therefore, we concluded that the addition of 10% CM derived from cAMSC in the freezing medium protected dog sperm during the cryopreservation process.

Characterization of viability, mitochondrial activity, acrosomal integrity and capacitation status in boar sperm during in vitro storage at different ambient temperatures

2002 ◽  
Vol 14 (8) ◽  
pp. 509 ◽  
Author(s):  
Li-Jun Huo ◽  
Kui-Zhong Yue ◽  
Zeng-Ming Yang
Keyword(s):  
Low Fertility ◽  
Blue Staining ◽  
Mitochondrial Activity ◽  
Hoechst 33258 ◽  
In Vitro Storage ◽  
Coomassie Blue Staining ◽  
Boar Sperm ◽  
Boar Semen ◽  
Acrosome Integrity

Extended storage of unfrozen boar semen becomes an alternative because the use of frozen–thawed boar sperm results in low fertility. Sperm viability, mitochondrial activity, capacitation and acrosome integrity of freshly ejaculated boar semen stored in vitro for up to 48 h at 4°C, 15°C, 20°C and 39°C was characterized during the study. The viability of boar sperm was assessed by both Hoechst 33258 and SYBR-14/PI staining. Mitochondrial function was assessed by JC-1 staining. Capacitation status was determined by chlortetracycline (CTC)/Hoechst 33258 staining. The acrosome integrity was analysed with Coomassie blue staining. These data were derived from three ejaculates each from three crossbred boars. The viabilities assessed with SYBR-14/PI, Hoechst 33258 and JC-1 staining correlated highly (r > 0.980). In freshly ejaculated boar semen, 96 ± 1% of the sperm did not take up the Hoechst 33258, whereas 95 ± 2% were stained by SYBR-14 and 96 ± 2% of the sperm had mitochondria exhibiting positive JC-1 staining. Staining with CTC/Hoechst 33258 suggested that a high percentage of sperm became capacitated after 24 h storage at 15°C and 20°C. There were 62 ± 2% (15°C) and 89�±�2% (20°C) capacitated sperm by 48 h. Moreover, most of the capacitated sperm were acrosome intact. These results suggest that SYBR-14/PI, Hoechst 33258 or JC-1 staining can be used to effectively evaluate the quality of boar sperm during in vitro storage.

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