How to select ICSI-viable sperm from the most challenging samples

2021 ◽  
Author(s):  
Reza Nosrati ◽  
David Sinton
Keyword(s):  
Viable Sperm

Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability

Reproduction ◽  
2000 ◽  
pp. 145-152 ◽  
Author(s):  
B Pintado ◽  
J de la Fuente ◽  
ER Roldan
Keyword(s):  
Nucleic Acid ◽  
Propidium Iodide ◽  
Hoechst 33258 ◽  
Experimental Conditions ◽  
Functional Studies ◽  
Viable Cells ◽  
Direct Microscopic Examination ◽  
Acrosomal Exocytosis ◽  
Viable Sperm ◽  
Boar Spermatozoa

This study was designed to assess whether nucleic acid stains such as propidium iodide and Hoechst 33258 and the cytosolic stain eosin identified equivalent proportions of non-viable cells. Sub-samples of boar spermatozoa stored for up to 72 h, and frozen bull spermatozoa stored in straws and thawed before staining, were exposed to either propidium iodide or Hoechst 33258 alone or in combination. Additional sub-samples were stained with eosin-nigrosin and subsequently with Giemsa. The proportion of non-viable cells identified by propidium iodide alone was equivalent to that observed when it was used in combination with the other fluorescent probe. Similar results were observed for Hoechst 33258. However, direct microscopic examination of sub-samples exposed to both stains revealed that a proportion of spermatozoa stained with propidium iodide did not incorporate Hoechst 33258. This was found consistently in boar and bull spermatozoa under the different experimental conditions used. Quantification showed that the proportion of propidium iodide-positive cells was significantly higher than Hoechst 33258-positive cells. Furthermore, the proportion of propidium iodide-positive cells was higher than cells stained with eosin, but no differences were found between the number of cells stained with Hoechst 33258 or eosin. The proportion of cells stained with propidium iodide was positively correlated with the proportion stained with either Hoechst 33258 or eosin, despite the observation that more cells incorporated propidium iodide. Taken together, these results indicate that there are differences in the ability of fluorescent probes to identify non-viable sperm cells and that this should be considered when staining protocols are used to analyse sperm viability, or when viability is used as a discriminating factor in functional studies, such as those related to acrosomal exocytosis.

scholarly journals Reproductive Anatomy of Chondrichthyans: Notes on Specimen Handling and Sperm Extraction. II. Sharks and Chimaeras

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2191
Author(s):  
Pablo García-Salinas ◽  
Victor Gallego ◽  
Juan F. Asturiano
Keyword(s):  
Artificial Insemination ◽  
Captive Breeding ◽  
Ex Situ ◽  
Specimen Handling ◽  
Viable Sperm ◽  
Abdominal Massage ◽  
Reproductive Anatomy ◽  
Conservation Projects ◽  
Breeding Techniques

The chondrichthyan fishes, which comprise sharks, rays, and chimaeras, are one of the most threatened groups of vertebrates on the planet. Given this situation, an additional strategy for the protection of these species could be the ex situ conservation projects developed in public aquaria and research centers. Nevertheless, to increase sustainability and to develop properly in situ reintroduction strategies, captive breeding techniques, such as sperm extraction and artificial insemination, should be developed. These techniques are commonly used in other threatened species and could be also used in chondrichthyans. However, the different reproductive morphologies found in this group can complicate both processes. Therefore, a comparison of the reproductive anatomy of eight distinct chondrichthyans, with an emphasis on those important differences when performing sperm extraction or artificial insemination, is carried out herein. Sharks and chimaeras belonging to the Scyliorhinidae, Carcharhinidae, Centrophoridae, Etmopteridae, Hexanchidae, and Chimaeridae families were obtained from commercial fisheries, public aquaria, and stranding events. In addition, the process of obtaining viable sperm samples through cannulation, abdominal massage, and oviducal gland extraction is described in detail for both living and dead animals.

Isolation and Characterization of Viable Sperm Cells from Tricellular Pollen Grains

1988 ◽  
pp. 245-250 ◽  
Author(s):  
E. Matthys-Rochon ◽  
S. Detchepare ◽  
V. Wagner ◽  
P. Roeckel ◽  
C. Dumas
Keyword(s):  
Pollen Grains ◽  
Sperm Cells ◽  
Isolation And Characterization ◽  
Viable Sperm

35 EFFECTS OF THAWING TEMPERATURE OF FROZEN SEMEN ON VIABILITY OF REFROZEN AND THAWED CHICKSO (KOREAN BRINDLE CATTLE) AND KOREAN ALBINO CATTLE SPERMATOZOA

2016 ◽  
Vol 28 (2) ◽  
pp. 147
Author(s):  
S. W. Kim ◽  
C. Y. Choe ◽  
D. K. Kim ◽  
A. R. Choi ◽  
H. H. Seong
Keyword(s):  
Genetic Resources ◽  
Sperm Concentration ◽  
Cooling Rates ◽  
Mean Values ◽  
Freeze Thaw ◽  
Frozen Semen ◽  
Native Cattle ◽  
Viable Sperm ◽  
The Mean ◽  
Thawing Procedure

Germplasm cryopreservation from a desired species with agricultural and genetic importance would protect them from the risk for extinction. Semen freezing from Korean native cattle would be a good approach for protecting genetic resources due to their limited numbers. It has been known that sperm could resist cryo-damages by freeze-thaw cycles. Thus, we performed 2 refreezing experiments with different initial thawing temperatures using frozen Korean native cattle semen. A total of 5 Hanwoo, Korean Albino, and brindle cattle were used as semen donors. After thawing by using 5°C/2 min or 37°C/40 s with cooling rates, the semen was diluted with the same volume of cryo-media in the first thawing temperature and refrozen. Sperm motilities were determined and compared between animals and groups after rethawing. The mean sperm concentration and motility was 45 × 106 mL–1 (range 2.3 to 89 × 106 mL–1) and 40% (range 13 to 55%). Mean values of motility and viability of sperm that underwent second preservation were significantly higher in 5°C than in 37°C (P < 0.01). However, the activity of viable sperm thawed at 5°C was significantly decreased before refreezing. It is estimated that refreezing of frozen semen from rare Korean native cattle is possible with resistant properties of survived spermatozoa. The higher motility and viability of refrozen semen could be obtained with 5°C thawing procedure for reuse of frozen semen.

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 Initial collection, characterization, and storage of tuatara (Sphenodon punctatus) sperm offers insight into their unique reproductive system

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0253628
Author(s):  
Sarah K. Lamar ◽  
Nicola J. Nelson ◽  
Jennifer A. Moore ◽  
Helen R. Taylor ◽  
Susan N. Keall ◽  
...  
Keyword(s):  
Sperm Cell ◽  
Sperm Cells ◽  
Motile Sperm ◽  
Sphenodon Punctatus ◽  
Viable Sperm ◽  
First Time ◽  
And Storage ◽  
Very High ◽  
Insight Into ◽  
Scanning Electron

Successful reproduction is critical to the persistence of at-risk species; however, reproductive characteristics are understudied in many wild species. New Zealand’s endemic tuatara (Sphenodon punctatus), the sole surviving member of the reptile order Rhynchocephalia, is restricted to 10% of its historic range. To complement ongoing conservation efforts, we collected and characterized mature sperm from male tuatara for the first time. Semen collected both during mating and from urine after courting contained motile sperm and had the potential for a very high percentage of viable sperm cells (98%). Scanning electron microscopy revealed a filiform sperm cell with distinct divisions: head, midpiece, tail, and reduced end piece. Finally, our initial curvilinear velocity estimates for tuatara sperm are 2–4 times faster than any previously studied reptile. Further work is needed to examine these trends at a larger scale; however, this research provides valuable information regarding reproduction in this basal reptile.

scholarly journals Conservation of wild silkworm genetic resources through cryopreservation: Standardization of sperm processing

2018 ◽  
Vol 10 (2) ◽  
pp. 544-547
Author(s):  
G. Lokesh ◽  
Geetha N Murthy ◽  
Veeranna Gowda ◽  
Alok Sahay ◽  
Gargi Gargi
Keyword(s):  
Genetic Resources ◽  
Seminal Vesicle ◽  
Artificial Insemination ◽  
Biologically Active ◽  
Ex Situ ◽  
Female Moth ◽  
Male Moth ◽  
Semen Collection ◽  
Wild Silkworm ◽  
Viable Sperm

Conservation of the invaluable sericigenous genetic resources is of prime importance with respect to their utilization and improvement for wider exploitation. Conservation of wild silkworms and its applicability in hybridization have limitations due to incompatibility, less amenability, change of behaviour under ex situ conditions, non-synchronization of moth eclosion and difficulties in mating between variables. In view of this, the newer technologies such as cryopreservation and artificial insemination are offering better strategies for preservation of biologically active samples like semen at sub-zero temperature (-196º C) conditions for longer duration. In this context, under standardization of sperms preservation from wild silkworms, two methods of semen collection were scrutinized for obtaining active and viable sperm for cryopreservation and further artificial insemination. Semen collection from the seminal vesicle of freshly emerged male moth and the other from the bursa copulatrix (BC) and spermatheca of the female moth after mating. The sperms in the semen collected from seminal vesicle are in the form of bundles known as eupyrene sperm bundles and apyrene sperms. The morphology and behaviour of these sperm bundles were recorded through microscopic examination. To study the density and motility behaviour of the sperms, sperm bundles were treated with proteolytic enzyme (~2-3µg/ml) to digest the membrane and release the sperms. The density and motility behaviour of sperms in the semen recovered from the BC and spermatheca of female moth after mating were higher compared to those released after digestion of sperm bundles from seminal vesicle of the male moth.

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