Initial Characterization of Male Southern Stingray (Hypanus americanus) Reproductive Parameters and Preliminary Investigation of Sperm Cryopreservation

This study investigated the reproductive biology and sperm cryopreservation of ex situ southern stingrays (Hypanus americanus) by semen collection and characterization and the development and validation of an enzyme-linked immunoassay for plasma total testosterone. Semen was collected in March and June using a manual massage technique, and the ejaculates were assessed for volume, pH, osmolarity, motility, status (0–5 scale: 0 = no forward progression, 5 = rapid linear progression) and total sperm count. Semen was extended in Hank’s elasmobranch ringer solution containing 10% DMSO, 10% glycerol or 5% glycerol with 5% N-methylformamide and cryopreserved using a conventional freezing method (~−50 °C/min) or a modified slow freezing method (~−3 °C/min). Body condition was scored from 1–5 and was noted to be low in March (1.93 ± 0.07) due to feeding practices and increased by June (2.93 ± 0.05) after dietary corrections were made. A concomitant increase (p < 0.05) in plasma total testosterone concentration and sperm motility was noted between March (8.0 ± 7.2 ng/mL, 5.71 ± 2.77%) and June (97.3 ± 11.3 ng/mL, 51.4 ± 14.3%). Samples cryopreserved using a modified slow freeze method (~−3 °C/min) had higher post-thaw motility and plasma membrane integrity than conventionally cryopreserved samples. Data indicate that southern stingray sperm morphometrics adheres to those of other elasmobranch species and that a slow cooling rate may be an avenue of research to improve southern stingray sperm survival during cryopreservation.

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Development of Sperm Cryopreservation Protocols for Sharks and Rays: New Tools for Elasmobranch Conservation

Frontiers in Marine Science ◽

10.3389/fmars.2021.689089 ◽

2021 ◽

Vol 8 ◽

Author(s):

Pablo García-Salinas ◽

Victor Gallego ◽

Juan F. Asturiano

Keyword(s):

Sperm Quality ◽

Membrane Integrity ◽

Egg Yolk ◽

Ex Situ ◽

Conservation Strategies ◽

Sperm Cryopreservation ◽

In Captivity ◽

Reproductive Techniques ◽

Elasmobranch Species ◽

Reproductive Cycles

Elasmobranchs are one of the most endangered vertebrate groups on the planet, but despite this situation the use of reproductive techniques in elasmobranch conservation strategies has been scarce. Among these techniques, sperm preservation is a potential tool for ex situ conservation and aquaria sustainability. However, there are no widespread preservation protocols for elasmobranch sperm, and shark sperm cryopreservation has never been achieved before. Here we present the establishment of successful cryopreservation protocols for elasmobranch sperm, tested in several species. We have formulated a sperm extender that can be used for different elasmobranch species, capable of maintaining sperm motility for several weeks. Additionally, we achieved the cryopreservation of sperm by previously diluting it in our extender and supplementing it with different combinations of cryoprotectants. The effects of methanol and dimethyl sulfoxide as permeating cryoprotectants were evaluated, as well egg yolk as a non-permeating cryoprotectant. Sperm quality was assessed by studying the motility and membrane integrity post-thawing, demonstrating its effectiveness in the 10 species tested, including two which are considered Critically Endangered. This is the first time that shark sperm cryopreservation has been reported, broadening our knowledge of the reproductive techniques that can be applied to elasmobranchs and laying the foundations for the first cryobanks for shark and ray sperm. Outcomes from this study will be useful for ex situ conservation efforts developed by public aquaria. A regular supply of frozen sperm will reduce the problems that result from the transport of specimens, inbreeding or lack of synchronized reproductive cycles in captivity.

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Evaluation of motility, membrane status and DNA integrity of frozen–thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation

Reproduction ◽

10.1530/rep-11-0490 ◽

2012 ◽

Vol 143 (6) ◽

pp. 799-813 ◽

Cited By ~ 15

Author(s):

G A Montano ◽

D C Kraemer ◽

C C Love ◽

T R Robeck ◽

J K O'Brien

Keyword(s):

Bottlenose Dolphin ◽

Sperm Quality ◽

Critical Evaluation ◽

Membrane Integrity ◽

Ex Situ ◽

Sperm Chromatin ◽

Dna Integrity ◽

Freezing Method ◽

Frozen Semen ◽

Motility Parameters

Artificial insemination (AI) with sex-sorted frozen–thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen–thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen–thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze–thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

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Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa

Reproduction Fertility and Development ◽

10.1071/rd16190 ◽

2017 ◽

Vol 29 (8) ◽

pp. 1556 ◽

Cited By ~ 8

Author(s):

S. Morrow ◽

J. Gosálvez ◽

C. López-Fernández ◽

F. Arroyo ◽

W. V. Holt ◽

...

Keyword(s):

Xenopus Laevis ◽

Membrane Damage ◽

Membrane Integrity ◽

Subsequent Development ◽

Xenopus Tropicalis ◽

Model Organisms ◽

Sperm Chromatin ◽

Dna Integrity ◽

Sperm Cryopreservation ◽

Sperm Plasma Membrane

There is growing concern over the effect of sperm cryopreservation on DNA integrity and the subsequent development of offspring generated from this cryopreserved material. In the present study, membrane integrity and DNA stability of Xenopus laevis and Xenopus tropicalis spermatozoa were evaluated in response to cryopreservation with or without activation, a process that happens upon exposure to water to spermatozoa of some aquatic species. A dye exclusion assay revealed that sperm plasma membrane integrity in both species decreased after freezing, more so for X. laevis than X. tropicalis spermatozoa. The sperm chromatin dispersion (SCD) test showed that for both X. tropicalis and X. laevis, activated frozen spermatozoa produced the highest levels of DNA fragmentation compared with all fresh samples and frozen non-activated samples (P < 0.05). Understanding the nature of DNA and membrane damage that occurs in cryopreserved spermatozoa from Xenopus species represents the first step in exploiting these powerful model organisms to understand the developmental consequences of fertilising with cryopreservation-damaged spermatozoa.

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Quality of ejaculate of male patients with type 2 diabetes mellitus (DM), vaccinated with GamCovidVac (Sputnik V)

Diabetes Mellitus ◽

10.14341/dm12775 ◽

2022 ◽

Vol 24 (5) ◽

pp. 422-426

Author(s):

D. I. Esaulenko ◽

R. R. Rozhivanov ◽

V. V. Shishkina

Keyword(s):

Diabetes Mellitus ◽

Type 2 Diabetes ◽

Type 2 Diabetes Mellitus ◽

Testosterone Level ◽

Glycated Hemoglobin ◽

Sperm Count ◽

Total Testosterone ◽

Male Patients

Background: New coronavirus infection (Covid-19) in patients with diabetes type 2 mellitus (DM) often has severe clinical course and manifestation. This comorbidity is a reasonable indication for vaccination. Male patients are often concerned about the vaccination impact on their fertility, so the current research of this issue seems to be essential and relevant.Aims: To evaluate the quality of ejaculate in type 2 diabetes mellitus (DM) patients, vaccinated by GamCovidVac (Sputnik V).Materials and Methods: The pilot observational prospective study included 30 males with type 2 diabetes mellitus (DM). The study continued from February 2021 till June 2021. The research design involved medical history analysis, glycated hemoglobin (HbA1c) tests, total testosterone level in blood measurement, semen analysis (sperm count test). Group comparison was performed by Wilcoxon Signed Ranks Test. The differences were considered statistically significant at p<0.05.Results: After vaccination 19 patients (63%) demonstrated a temperature rise which lasted for 2 days; 26 patients (87%) complained of tenderness in the injections site which lasted up to 5 days. Though a few patients reported general somatic side effects after the vaccination, there have been no statistically significant deviations in sperm count, viability, function and morphology. The levels of glycated hemoglobin and total testosterone remained unchanged.Conclusion: The study revealed no negative impact of GamCovidVac on ejaculate quality, total testosterone level and compensation of carbohydrate metabolism.

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

Reproduction Fertility and Development ◽

10.1071/rd08036 ◽

2008 ◽

Vol 20 (6) ◽

pp. 724 ◽

Cited By ~ 21

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.

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Relationship between semen quality and seminal plasma cholesterol, triacylglycerols and proteins in the domestic cat

Journal of Feline Medicine and Surgery ◽

10.1177/1098612x19889072 ◽

2019 ◽

Vol 22 (10) ◽

pp. 882-889 ◽

Cited By ~ 1

Author(s):

María F García ◽

Romina Nuñez Favre ◽

María C Stornelli ◽

Ramiro Rearte ◽

María C García Mitacek ◽

...

Keyword(s):

Seminal Plasma ◽

Sperm Morphology ◽

Semen Quality ◽

Sperm Count ◽

Sodium Dodecyl ◽

Sperm Concentration ◽

Membrane Integrity ◽

Domestic Cat ◽

Sds Page ◽

The Relationship

Objectives The current study aimed to evaluate the relationship between specific seminal plasma components – cholesterol (CHOL), triacylglycerols (TAG) and total protein (PROT) concentrations – and semen quality in cats. A further aim was to determine the relationship between specific seminal protein bands and semen quality. Methods Thirteen toms, 2–5 years of age, were included. Semen collection was performed by electroejaculation every 4 weeks. Fifty-eight ejaculates were assessed for motility, velocity, volume, sperm concentration, total sperm count, viability, acrosome integrity, plasma membrane integrity and sperm morphology. Samples were divided into two groups: good semen quality (GSQ) and poor semen quality (PSQ). After evaluation, seminal plasma was separated from the sperm by centrifugation and stored at −20°C. CHOL, TAG and PROT concentrations were then assessed and seminal plasma protein profile was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results Seminal plasma CHOL and TAG concentrations, motility, velocity, sperm concentration, total sperm count and sperm morphology were significantly higher in GSQ cats compared with PSQ cats ( P <0.01). Moreover, seminal plasma SDS-PAGE analysis showed an identifiable extra band exclusively in the GSQ group. Conclusions and relevance Data obtained in this study showed that seminal plasma CHOL and TAG concentrations and specific protein bands could be used to improve semen evaluation in toms. In this sense, the 14 kDa protein band could be a valuable marker for semen quality in the cat and should be further investigated. However, more studies are necessary to determine its relationship with fertility.

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

Animals ◽

10.3390/ani10101899 ◽

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.

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Assessment of basic seminal characteristics, sperm cryopreservation and heterologous in vitro fertilisation in the fishing cat (Prionailurus viverrinus)

Reproduction Fertility and Development ◽

10.1071/rd05098 ◽

2006 ◽

Vol 18 (3) ◽

pp. 373 ◽

Cited By ~ 29

Author(s):

Khongsak Thiangtum ◽

William F. Swanson ◽

JoGayle Howard ◽

Wanchai Tunwattana ◽

Dakara Tongthainan ◽

...

Keyword(s):

Sperm Motility ◽

Sperm Morphology ◽

Domestic Cat ◽

Ex Situ ◽

In Vitro Fertilisation ◽

Genetic Management ◽

Slow Cooling ◽

Breeding Programmes ◽

Acrosomal Integrity

Conservation of the fishing cat, a threatened south-east Asian felid, could benefit from effective ex situ genetic management and breeding programmes, including the use of assisted reproduction. The aims of the present study were to: (1) characterise basal seminal traits of fishing cats in Thailand zoos; and (2) investigate the effect of cryopreservation on sperm motility, acrosomal integrity and in vitro function. Seminal traits were evaluated in electroejaculates collected from eight males. Spermatozoa were diluted in n-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid Tris (TEST)-yolk buffer (TYB) without glycerol, then diluted further with TYB with glycerol (4% final concentration) at either 25°C or after slow cooling to 5°C and frozen in straws over liquid nitrogen vapour. After thawing, sperm function was assessed by insemination of viable domestic cat oocytes. Fishing cat ejaculates averaged (± s.e.m.) 43.6 ± 14.2 × 106 motile spermatozoa with 33.5 ± 6.8% normal sperm morphology. Semen processing had a negligible effect (P > 0.05) on sperm motility and acrosomal integrity, but values were reduced (P < 0.05) after thawing. All thawed samples fertilised domestic cat oocytes, with 62.1% (36/58) of mature oocytes cleaving. Glycerol addition at 5°C resulted in higher (P < 0.05) post-thaw motility and intact acrosomes than glycerol addition at 25°C. In conclusion, good-quality ejaculates can be obtained from Thai fishing cats and their spermatozoa exhibit adequate function after cryopreservation for in vitro fertilisation procedures.

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Development of sperm cryopreservation protocol of endangered spiny eel, Mastacembelus armatus (Lacepede 1800) for ex-situ conservation

Cryobiology ◽

10.1016/j.cryobiol.2016.10.004 ◽

2016 ◽

Vol 73 (3) ◽

pp. 316-323 ◽

Cited By ~ 9

Author(s):

M. Mahfujur Rahman ◽

M. Rahmat Ali ◽

M. Rafiqul Islam Sarder ◽

M. Fazlul Awal Mollah ◽

Najmus Sakib Khan

Keyword(s):

Ex Situ Conservation ◽

Ex Situ ◽

Sperm Cryopreservation ◽

Mastacembelus Armatus ◽

Cryopreservation Protocol

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Slow cooling prevents cold-induced damage to sperm motility and acrosomal integrity in the black-footed ferret (Mustela nigripes)

Reproduction Fertility and Development ◽

10.1071/rd06096 ◽

2007 ◽

Vol 19 (5) ◽

pp. 652 ◽

Cited By ~ 18

Author(s):

R. M. Santymire ◽

P. E. Marinari ◽

J. S. Kreeger ◽

D. E. Wildt ◽

J. G. Howard

Keyword(s):

Sperm Motility ◽

Seminal Plasma ◽

Artificial Insemination ◽

Sperm Cryopreservation ◽

Slow Cooling ◽

Rapid Cooling ◽

Mustela Nigripes ◽

Factors Influencing ◽

Rate Of Cooling ◽

Acrosomal Integrity

The endangered black-footed ferret (Mustela nigripes) has benefited from artificial insemination; however, improved sperm cryopreservation protocols are still needed. The present study focused on identifying factors influencing gamete survival during processing before cryopreservation, including: (1) the presence or absence of seminal plasma; (2) temperature (25°C v. 37°C); (3) type of medium (Ham’s F10 medium v. TEST yolk buffer [TYB]); (4) cooling rate (slow, rapid and ultra-rapid); and (5) the presence or absence of glycerol. Seminal plasma did not compromise (P > 0.05) sperm motility or acrosomal integrity. Sperm motility traits were maintained longer (P < 0.05) at 25°C than at 37°C in Ham’s or TYB, but temperature did not affect (P > 0.05) acrosomal integrity. Overall, TYB maintained optimal (P < 0.05) sperm motility compared with Ham’s medium, but Ham’s medium maintained more (P < 0.05) intact acrosomes than TYB. Slow cooling (0.2°C min–1) was optimal (P < 0.05) compared to rapid cooling (1°C min–1), and ultra-rapid cooling (9°C min–1) was found to be highly detrimental (P < 0.05). Results obtained in TYB with 0% or 4% glycerol were comparable (P > 0.05), indicating that 4% glycerol was non-toxic to ferret sperm; however, glycerol failed to ameliorate the detrimental effects of either rapid or ultra-rapid cooling. The results of the present study demonstrate that the damage observed to black-footed ferret spermatozoa is derived largely from the rate of cooling.

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