CryoLetters Volume 43 - Issue 3

Contents

CryoLetters 43 (3), 129-139 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22310110112

PERSPECTIVE: Recent advancements in vitrification cryodevices for gamete and gonadal tissue

Masrat-Un-Nisa1, Asloob Ahmad Malik*1, Khursheed Ahmad Sofi2, Arjuma Khatun1 and Nahida Yousuf1

  1. Division of Animal Reproduction, Gynaecology and Obstetrics, Faculty of Veterinary Science and Animal Husbandry, Shuhama, SKUAST-Kashmir, Srinagar, 190006, India
  2. Division of Veterinary Clinical Complex, Faculty of Veterinary Science and Animal Husbandry, Shuhama, SKUAST-Kashmir, Srinagar, 190006, India

Abstract

Cryopreservation of gametes and gonadal tissue is nowadays primarily accomplished through vitrification. Variables such as cooling rate, viscosity and volume of vitrification solution are critical in gamete vitrification. In addition, sample size and stepwise exposure are also crucial for gonadal tissue vitrification. Recently a class of cryodevices has been developed to reduce the volume of vitrification solution so as to achieve higher cooling rates. Vitrification devices are classified as “open” or “closed” depending on whether the medium comes into direct contact with liquid nitrogen during the process. Examples of the open cryodevices for gamete vitrification are Cryotop, Cryolock, open pulled straw (OPS), etc., and closed devices are Vitrisafe, CryoTip, and high security vitrification kit. Similarly, for tissue vitrification open cryodevices used are needles, cryovials and closed devices used are Cryotissue, ovarian tissue cryosystem, etc. Among all the gamete cryodevices, Cryotop is unique and the best-selling micro-volume storage device. Use of this device has resulted in the highest number of babies born after embryo or oocyte vitrification. Another novel device, Kitasato vitrification system, is a vitrification solution absorber, which is similar to Cryotop but differs in one way, as it possesses a porous membrane that absorbs extra solution from the gamete. This review provides an update on the recent use of cryodevices for gamete and gonadal tissue vitrification.

Keywords: closed system; cryodevices; CryoLoop; CryoTip; Cryotop; Kitasato vitrification system

View PDF
Contents

CryoLetters 43 (3), 140-149 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22310110312

Influence of sperm post-thaw subpopulations of Angus and Nelore bulls on pregnancy rates by fixed-time artificial insemination

Myrian Megumy Tsunokawa Hidalgo1, Ana Beatriz Marques de Almeida1, Fábio Lucas Zito de Moraes2, Rodrigo Yudi Palhaci Marubayashi3, Thales Ricardo Rigo Barreiros4 and Maria Isabel Mello Martins1*

  1. LARAA, Universidade Estadual de Londrina- UEL, Brazil
  2. Veterinário Autônomo, Londrina, Brazil
  3. Departamento de pós graduação em Agronomia, Universidade Estadual de Londrina- UEL, Brazil
  4. Laboratório de Reprodução Animal, Universidade Estadual do Norte do Parana, Bandeirantes, Brazil

Abstract

Background

The heterogeneity of ejaculate indicates that fertility is still variable among bulls and that more stringent evaluation methods are needed to identify the ejaculates suitable for AI.

Objective

To identify and characterize the sperm subpopulations (SP) in thawed semen doses of Nelore and Angus bulls and to evaluate the influence of these sperm subpopulations on pregnancy rate in cows submitted to fixed-time AI (FTAI).

Materials and methods

A dose of post-thawed semen from each bull (n=18; consisting of Angus n = 9 and Nelore n = 9) was analyzed for: sperm kinetics; morphology and plasma membrane integrity; and the determination of the sperm subpopulations. Differences between the groups were estimated with the t-test considering a significance level of <5%.

Results

There was no influence between breeding bulls for sperm morphology, plasma membrane integrity, and pregnancy rate (P > 0.05). Regarding the kinetic parameters evaluated by the CASA system, Nelore had greater values, for cells with slow velocity (Angus: 16.4 %; Nelore: 21.74%; P = 0.028). In contrast, ANGUS bulls had more static cells (Angus: 27.2%; Nelore: 9.3%; P = 0.048). Based on CASA system data and clustering procedures, four sperm subpopulations were statistically established. In Angus bulls, a higher level of fast and nonlinear spermatozoa were found in SP3 (33.3%), followed by SP1 (32.7%) with fast and progressive spermatozoa. Whereas, SP1 of Nelore bulls had 33.8% fast and progressive spermatozoa, followed by 32.2% of SP3 with fast and nonlinear spermatozoa.

Conclusion

Both breeds of bulls presented similar proportions of sperm SP. Consequently, no influence on the pregnancy rates was shown in cows submitted to the IATF programs on a large scale.

Keywords: bulls; CASA system; fertility; FTAI, semen

Contents

CryoLetters 43 (3), 150-157 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22310110512

Effects of unconjugated gold, silver and titanium dioxide nanoparticles on bovine spermatozoa at various stages of cryopreservation

Megha Pande1*, Shrikant Tyagi1*, Suresh Kumar2, Y.K. Soni3, N. Chand1, A.S. Sirohi1, Sarika1, I. Devi4 and S. Mahajan1

  1. Semen Freezing Laboratory, Division of Cattle Physiology and Reproduction, ICAR-Central Institute for Research on Cattle, Meerut, Uttar Pradesh, India
  2. Animal Physiology Laboratory, Division of Cattle Physiology and Reproduction, ICAR-Central Institute for Research on Cattle, Meerut, Uttar Pradesh, India
  3. Animal Physiology and Reproduction Section, ICAR-Central Institute for Research on Goats (ICAR-CIRG), Makhdoom, Farah, Uttar Pradesh, India
  4. Division of Cattle Nutrition and Management, ICAR-Central Institute for Research on Cattle, Meerut, Uttar Pradesh, India

Abstract

Background

The increasing use of nanoparticles (NP) for gender-selected spermatozoa, sperm-enriched semen and novel extenders raises the concern of undesirable effects on fertility and sperm function.

Objective

To investigate the effects of gold (Au-), silver (Ag-), and titanium dioxide (TiO2-) NPs on the motility and sperm functions in bovine spermatozoa at various stages of cryopreservation.

Materials and methods

Frieswal (Sahiwal × Holstein Friesian) bull semen ejaculates (N = 24) were challenged with unconjugated and ligand-free Au-, Ag-, and TiO2-NPs.

Results

At post-dilution (fresh) stage, there was no significant difference observed in progressive motility and viability amongst the control and any nanoparticle-treated groups, though plasma membrane integrity was significantly reduced in nanoparticle-treated groups (p < 0.05). The acrosome intactness was also significantly reduced in the groups of Ag-NP and TiO2-NP (p < 0.05), while there was no effect observed in the Au-NP group. At post-equilibration stage, a significant reduction in motility, viability, and plasma membrane integrity was observed in all three nanoparticle-treated groups (p < 0.05). There was no difference in intact acrosome between the control and Au-NPs groups; which was significantly higher than the Ag-NP and TiO2-NP groups (p < 0.05). At post-thaw stage, all NP groups resulted in a significant reduction of motility, viability, acrosome intactness and plasma membrane integrity (p< 0.05). Besides, TiO2-NPs appear to be significant more toxic (p< 0.05) among three NP groups, and Au-NPs appear to be lesser toxic.

Conclusion

Bovine spermatozoa are adversely affected by Au-, Ag- and TiO2-NPs that may impair sperm motility and other functions.

Keywords: Frieswal bull; gold; nanotoxicity; silver; spermatozoa; titanium dioxide

Contents

CryoLetters 43 (3), 91-98 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22310110712

Correlation between dissolved oxygen level, antioxidants and oxidants in semen diluted with partially deoxygenated extender at various stages of cryopreservation

B. Balamurugan1, S.K. Ghosh2, S.A. Lone3*, J.K. Prasad2, M. Ramamoorthy2 and A. Kumar4

  1. Department of Veterinary Gyneacology and Obstetrics, BHU-Faculty of Veterinary and Animal Science, RGSC, Barkachha, Uttar Pradesh-231001, India
  2. Division of Animal Reproduction, ICAR-Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, 243122, India
  3. Animal Husbandry Department, Government of Jammu & Kashmir, India
  4. Biochemistry Division, ICAR-Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, 243122, India

Abstract

Background

Dissolved oxygen (DO) in semen dilutor may lead to the production of reactive oxygen species (ROS) and buffalo sperm may become more prone to deleterious effects of ROS due to the presence of high amounts of polyunsaturated fatty acids (PUFAs) in their membranes.

Objective

To study the correlation between dissolved oxygen level, antioxidants and oxidants in semen diluted with partially deoxygenated extender at various stages of cryopreservation.

Materials and methods

Each semen sample was split into two aliquots viz., Aliquot I [diluted with Extender I (control: without deoxygenation)] and Aliquot II [diluted with Extender II: partially deoxygenated by liquid nitrogen (LN) flushing], which were diluted, filled in straws, cryopreserved and evaluated post-thaw.

Results

The DO levels (P < 0.05) decreased significantly after LN flushing of the extender and they increased significantly (P < 0.05) in post-thaw semen. The progressive motility, viability, hypo-osmotic swelling response, acrosomal integrity, glutathione peroxidase (GPx), total antioxidant capacity (TAC) and superoxide dismutase (SOD) decreased significantly (P < 0.05) in both control and treated semen after thawing. SOD and TAC were positively correlated in semen treated with normal extender at the pre-freeze stage; however, in semen treated with partially deoxygenated extender, no correlation was found between SOD and TAC at the pre-freeze stage. ROS and total TAC were negatively correlated in semen treated with partially deoxygenated extender at the pre-freeze stage; however, no correlation was found between ROS and TAC in control semen.

Conclusion

The partial deoxygenation of extender affects the correlation between sperm quality parameters, antioxidants, and oxidants during different stages of semen cryopreservation.

Keywords: antioxidants; cryopreservation; deoxygenation; extender; oxidants

Contents

CryoLetters 43 (3), 167-174 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22310110212

Vitrification by transient vacuum flashing spray cooling of liquid nitrogen

Fengmin Su*, Yiming Fan, Chi Zhang, Yifan Wang, Yanyang Wang and Benli Peng

  • Institute of Marine Engineering and Thermal Science, Dalian Maritime University, Dalian, China

Abstract

Background

The transient vacuum flashing spray cooling of liquid nitrogen (LN2) on a microstructured surface can provide ultra-fast cooling rate and may improve cell survival rates.

Objective

To utilize flashing spray cooling of LN2 instead of film boiling to improve further cell vitrification. METHOD: This study analyzed the effects of the three key parameters (flow rate of liquid nitrogen, ambient pressure, and spray distance) on the cooling process by experimentation.

Methods

This study analyzed the effects of the three key parameters (flow rate of liquid nitrogen, ambient pressure, and spray distance) on the cooling process by experimentation.

Results

The experimental results showed that the vacuum flashing spray cooling of LN2 can gain higher cooling rates than that achieved by film boiling in conventional vitrification methods. The three parameters all affected the vacuum flash evaporation spray cooling of LN2, and their effect trends were not monotonous but followed a parabolic trend that increased and then decreased. That is, the three parameters all have optimum values to the cooling process.

Conclusion

Vacuum flash evaporation spray cooling can develop the ultra-fast cooling rates needed to enhance cell vitrification.

Keywords: cell cryopreservation; flashing spray cooling; liquid nitrogen; transient heat transfer

Contents

CryoLetters 43 (3), 110-119 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22310110412

Cryopreservation of HEP-G2 cells attached to substrates: the benefit of sucrose and trehalose in combination with dimethyl sulfoxide

Bumbat Myagmarjav and Baolin Liu*

  • Institute of Biomedical Technology, The University of Shanghai for Science and Technology, 516 Jun Gong Road, Shanghai, 200093, China

Abstract

Background

Cryopreservation of mammalian cells is mainly done in cryovials as free cell suspension in 5 to 10% (v/v) dimethyl sulfoxide (DMSO). Relatively little attention has been paid to cryopreservation of adherent cell monolayers.

Objective

To investigate the appropriate cryoprotectant (CPA) formulations for the cryopreservation on HEP-G2 human tumor cells attached to the polystyrene plate and plastic surfaces.

Materials and methods

Five CPA formulations were evaluated for the cryopreservation of HEP-G2 cells attached to polystyrene plates and and plastic coverslips, using post-thaw cell viability as a performance indicator.

Results and conclusion

Hep-G2 cells attached to the plastic coverslips and polystyrene plate surfaces were successfully cryopreserved in 10% DMSO with sucrose and trehalose. The addition of saccharides enabled the reduction of DMSO concentration, replaced serum, and improved the functional capacity of post-thaw Hep-G2 cells. Cells attached to the plastic coverslips show significantly better results than those attached to the polystyrene plate surfaces after cryopreservation.

Keywords: attached HEP-G2 cells; cryopreservation; sucrose; trehalose

View PDF
Contents

CryoLetters 43 (3), 183-198 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22310110612

Beneficial effect of melatonin administration on the function of frozen-thawed rat testicular grafts

Jian-Min Zhang1, Xi-Lan Lu2, Hong-Xia Wang2 and Zi-Chao Liu1*

  1. Weifang Nursing Vocational College, Qingzhou, China
  2. Reproductive Medicine Center, Jinan Central Hospital, Jinan, China

Abstract

Background

Spermatogonia in testis is sensitive to the cytotoxicity of chemotherapy agents. Cryopreservation of testicular tissue may offer fertility restoration in young male cancer survivors.

Objective

To investigate the effect of melatonin on the survival of testicular grafts following cryopreservation and transplantation.

Materials and methods

Wister rats were randomly allocated into three groups: control group; saline group (cryopreservation + autograft + saline); and melatonin group (cryopreservation + autograft + melatonin). Malondialdehyde (MDA) content, glutathione peroxidase (GPx) activity and superoxide dismutase (SOD) activity were assessed on day 7 after autograft transplantation. At day 30, graft recovery, spermatogonia per round tubule, and serum testosterone concentration in grafts were measured.

Results

Melatonin significantly diminished MDA content, enhanced GPx and SOD activities. Furthermore, the recovery rate, number of spermatogonia per round tubule, and serum testosterone concentration in melatonin group was markedly higher than the saline group.

Conclusion

Melatonin administration at 20 mg/kg is effective in improving the function of frozen and thawed rat testicular graft. The protective role of melatonin can be attributed partly to the enhanced ROS scavenging and antioxidant enzyme activities.

Keywords: cryopreservation; melatonin; testis; transplantation