Concentrations of non-permeable cryoprotectants and equilibration temperatures are key factors for stallion sperm vitrification success

Hidalgo, M.; Consuegra, C.; Dorado, J.; Díaz-Jiménez, María; Ortiz, Isabel; Pereira, B.; Sánchez, Raúl; Crespo, F.

doi:10.1016/j.anireprosci.2018.06.022

Cited by 30 publications

(26 citation statements)

References 52 publications

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“…In humans, sucrose alone adequately preserves semen viability and motility [24]. Sucrose alone has also been demonstrated to support successful vitrification and ultimately fertilization in wild goats [23], mouflons [10], and stallions [25]. However, our results are in agreement with other studies in which some protein source is necessary to support survival during the initial cold shock of cryopreservation.…”

Section: Discussionsupporting

confidence: 90%

Vitrification Using Soy Lecithin and Sucrose: A New Way to Store the Sperm for the Preservation of Canine Reproductive Function

Pipan

Casal

Šterbenc

et al. 2020

Animals

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Simple Summary: Soy lecithin and sucrose were used at different concentrations to develop and compare different vitrification methods for the cryopreservation of canine semen. All of the sperm quality characteristics were effectively preserved after devitrification when vitrification extenders containing soy lecithin at 1% and 0.25 M sucrose were used. The results suggest that vitrification is effective, fast, and simple for cryopreservation of canine semen. Furthermore, the use of soy lecithin in lieu of animal proteins (e.g., egg yolk) facilitates semen shipping to countries with strict import requirements.Abstract: A challenge in freezing semen for short and long-term availability is avoiding damage to intact spermatozoa caused by the freezing process. Vitrification protocols provide better results through less manipulation of semen and shorter freezing time compared to slow freezing techniques. Our research was aimed at improving vitrification methods for canine semen. Semen quality was determined in 20 ejaculates after collection. Each ejaculate was divided into eight aliquots, each with a different extender. The control extender contained TRIS, citric acid, fructose, and antibiotics. Soy lecithin and sucrose were added to the control extender at different concentrations to make up the test extenders and final concentration of 50 × 10 6 spermatozoa/mL. From each group, a 33 µL (1.65 × 10 6 spermatozoa) suspension of spermatozoa was dropped directly into liquid nitrogen and devitrified at least one week later and evaluated as before. Soy lecithin at 1% and 0.25 M sucrose added to the base vitrification media effectively preserved all sperm qualities. Our results demonstrate the effectiveness of our methods. Vitrification media containing sucrose and soy lecithin cause a minimal decline in quality of canine semen after devitrification. Furthermore, extenders used in our research did not contain egg yolk, which was replaced by soy lecithin, thus allowing for ease of shipping to other countries with strict requirements.

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Section: Discussionsupporting

confidence: 90%

Vitrification Using Soy Lecithin and Sucrose: A New Way to Store the Sperm for the Preservation of Canine Reproductive Function

Pipan

Casal

Šterbenc

et al. 2020

Animals

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“…Our results showed the extender containing only non‐permeable agents was able to protect plasma membrane and DNA integrity in both groups, as no differences were found in comparison to extender containing glycerol. In the same way, a combination of sucrose and BSA resulted in high plasma membrane and DNA integrity percentages after stallion sperm cryopreservation in previous reports (Consuegra et al., ; Hidalgo et al., ). Discrepancy in motility results between fertile and subfertile groups regarding the use of glycerol might be explained because the effectiveness of non‐permeable cryoprotectants could be subjected to the initial sperm quality of the sample.…”

Section: Discussionsupporting

confidence: 67%

Is sperm cryopreservation in absence of permeable cryoprotectants suitable for subfertile donkeys?

Díaz-Jiménez

Dorado

Pereira

et al. 2019

Reprod Domestic Animals

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Sperm from fertile donkeys have been successfully frozen in absence of permeable cryoprotectants. The aim of this study was to determine whether this cryopreservation method is suitable for subfertile donkeys in comparison to conventional sperm freezing with glycerol. Ejaculates were collected from four Andalusian Donkeys: three fertile and one subfertile. Semen was frozen with an extender containing glycerol (GLY), or adding instead sucrose 0.25 molar and 1% bovine serum albumin (SUC) as non-permeable cryoprotectants. After thawing, samples were assessed for total (TM, %) and progressive (PM, %) sperm motility by CASA, plasma membrane integrity (PMI, %) by epifluorescence microscopy and DNA integrity (DFI, %) by SCSA. Results (mean ± SD) were compared between extenders in fertile and subfertile donkeys using the Student's t test. No differences between GLY and SUC treatments were found in the fertile group for the sperm parameters assessed. In subfertile donkey ejaculates, GLY resulted in significantly higher values than SUC for TM (25.5 ± 3.1 vs.19.6 ± 1.9) and PM (13.3 ± 5.1 vs. 4.0 ± 1.2), respectively. In conclusion, considering all the sperm parameters assessed, sperm freezing in absence of permeable cryoprotectants may not be still an option for cryopreservation of subfertile donkey sperm. K E Y W O R D S cryoprotectant, donkey, fertility, freezing, sperm | 103 DIAZ-JIMENEZ Et Al. | 105 DIAZ-JIMENEZ Et Al. ACK N OWLED G M ENTS The authors are grateful to 'Centro Rural Malpica' for providing animals and facilities. This study was funded by MINECO grant AGL2013-42726-R. M. Diaz-Jimenez is supported by a FPU fellowship from Spanish MECD ('Ministerio de Educación, Cultura y Deporte', Spain). CO N FLI C T O F I NTE R E S T None of the authors have any conflict of interest to declare. AUTH O R CO NTR I B UTI O N S M Hidalgo and M Diaz-Jimenez contributed to all sections. J Dorado and I Ortiz contributed to the study design, preparation and final approval of the manuscript. C Consuegra and B Pereira performed the experiments. All the authors were involved in revision and approval of the final version of the manuscript.

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“…The optimal concentration of CPAs seems to be species-specific and has been proposed as a key factor for sperm vitrification success, depending on the methodology used [24]. In a preliminary research, donkey sperm vitrified in straws showed significant higher sperm motility percentages when compared to vitrification in spheres [25].…”

Section: Introductionmentioning

confidence: 99%

“…It is a simple, fast, and cost-effective method to cryopreserve sperm, even under field conditions, since a reasonably equipped laboratory is required, making it attractive for the conservation of wild or endangered species and genetically valuable animals distributed in different regions, as it usually happens in endangered donkey breeds. Taking into account that sperm vitrification has led to similar or an increase in sperm quality after warming in comparison to conventional freezing in stallion [24], it could be considered to be another alternative to improve sperm cryopreservation in donkeys.…”

Section: Introductionmentioning

confidence: 99%

Vitrification of Donkey Sperm: Is It Better Using Permeable Cryoprotectants?

Hidalgo

Díaz-Jiménez

Consuegra

et al. 2020

Animals

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Vitrification by direct exposure of sperm to liquid nitrogen is increasing in popularity as an alternative to conventional freezing. In this study, the effect of permeable cryoprotectant agents for donkey sperm vitrification was compared to an extender containing non-permeable cryoprotectants. First, three different concentrations of sucrose (0.1, 0.2, and 0.3 molar, M) and bovine serum albumin, BSA (1, 5, and 10%) were compared. Secondly, the concentration of non-permeable agents producing the most desirable results was compared to an extender containing glycerol as permeable agent. Vitrification was performed by dropping 30 μL of sperm suspension directly into LN2 and warming at 42 °C. Sperm motility (total, TM; and progressive, PM) and plasma membrane integrity, PMI (mean ± SEM) were statistically compared between treatments. Sucrose 0.1 M showed a significantly higher percentage of total sperm motility (21.67 ± 9.22%) than sucrose 0.2 M (14.16 ± 4.50%) and 0.3 M (8.58 ± 6.22%); and no differences were found in comparison to the control (19.71 ± 10.16%). Vitrification with sucrose 0.1 M or BSA 5% obtained similar results for TM (21.67 ± 9.22% vs. 19.93 ± 9.93%), PM (13.42 ± 6.85% vs. 12.54 ± 6.37%) and PMI (40.90 ± 13.51% vs. 37.09 ± 14.28); but both showed higher percentages than glycerol (TM = 9.71 ± 4.19%; PM = 5.47 ± 3.17%; PMI = 28.48 ± 15.55%). In conclusion, donkey sperm vitrification in spheres using non-permeable cryoprotectants exhibited better sperm motility and viability parameters after warming than sperm vitrification using extenders containing permeable cryoprotectants.

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Concentrations of non-permeable cryoprotectants and equilibration temperatures are key factors for stallion sperm vitrification success

Cited by 30 publications

References 52 publications

Vitrification Using Soy Lecithin and Sucrose: A New Way to Store the Sperm for the Preservation of Canine Reproductive Function

Vitrification Using Soy Lecithin and Sucrose: A New Way to Store the Sperm for the Preservation of Canine Reproductive Function

Is sperm cryopreservation in absence of permeable cryoprotectants suitable for subfertile donkeys?

Vitrification of Donkey Sperm: Is It Better Using Permeable Cryoprotectants?

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