Cryoprotective Effect of Pentoxifylline on Spermatogonial Stem Cell During Transplantation into Azoospermic Torsion Mouse Model.

Preserving the spermatogonial stem cells (SSCs) in long periods of time during the treatment of male infertility using stem cell banking systems and transplantation is an important issue. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using 10 mM pentoxifylline (PTX) as an antioxidant in basal freezing medium. Testicular torsion-a mouse model for long-term infertility-was used to transplant fresh SSCs (n = 6), fresh SSCs treated with PTX (n = 6), cryopreserved SSCs with basal freezing medium (n = 6), and cryopreserved SSCs treated with PTX (n = 6). Eight weeks after germ cell transplantation, samples were assessed for proliferation, through evaluation of Ddx4 and Id4 markers, and differentiation via evaluation of C-Kit and Sycp3, Tnp1, Tnp2, and Prm1 markers. According to morphological and flow cytometry results, SSCs are able to form colonies and express Gfra1, Id4, α6-integrin, and ß1-integrin markers. We found positive influence from PTX on proliferative and differentiative markers in SSCs transplanted to azoospermic mice. In the recipient testis, donor SSCs formed spermatogenic colonies and sperm. Respecting these data, adding pentoxifylline is a practical way to precisely cryopreserve germ cells enriched for SSCs in cryopreservation, and this procedure could become an efficient method to restore fertility in a clinical setup. However, more studies are needed to ensure its safety in the long term.

The transfer temperature from slow cooling to cryogenic storage is critical for optimal recovery of cryopreserved mammalian cells.

Cryopreservation is a key step for the effective delivery of many cell therapies and for the maintenance of biological materials for research. The preservation process must be carefully controlled to ensure maximum, post-thaw recovery using cooling rates slow enough to allow time for cells to cryodehydrate sufficiently to avoid lethal intracellular ice. This study focuses on determining the temperature necessary at the end of controlled slow cooling before transfer to cryogenic storage which ensures optimal recovery of the processed cell samples. Using nucleated, mammalian cell lines derived from liver (HepG2), ovary (CHO) and bone tissue (MG63) this study has shown that cooling must be controlled to -40°C before transfer to long term storage to ensure optimal cell recovery. No further advantage was seen by controlling cooling to lower temperatures. These results are consistent with collected differential scanning calorimetry data, that indicated the cells underwent an intracellular, colloidal glass transition between -49 and -59°C (Tg'i) in the presence of the cryoprotective agent dimethyl sulfoxide (DMSO). The glass forms at the point of maximum cryodehydration and no further cellular dehydration is possible. At this point the risk of lethal intracellular ice forming on transfer to ultra-low temperature storage is eliminated. In practice it may not be necessary to continue slow cooling to below this temperature as optimal recovery at -40°C indicates that the cells have become sufficiently dehydrated to avoid further, significant damage when transferred into ultra-low temperature storage.

Application of Dimethyl Sulfoxide as a Therapeutic Agent and Drug Vehicle for Eye Diseases.

Dimethyl sulfoxide (DMSO) is an amphipathic molecule widely used as a solvent for water-insoluble substances, cryopreserving, and cell-biological therapies. It has known properties as an inducer of cellular differentiation, a free radical scavenger, and a radioprotectant. In addition, DMSO is used for its various therapeutic and pharmaceutical properties, such as anti-inflammatory, local and systemic analgesic, antibacterial, antifungal, antiviral, and membrane penetration enhancement agents. DMSO treatment can be given orally, intravenously, or topically for a wide range of indications. The administration of DMSO exhibits favorable outcomes in human eye diseases with low to none observed ocular or systemic ocular toxicity. Nevertheless, DMSO is an essential and nonpatentable potential therapeutic agent that remains underexplored and ignored by pharmaceutical developers and ophthalmologists. This current review takes data from experimental and clinical studies that have been published to substantiate the potential therapeutic efficacy of DMSO and stimulate the research of its application in clinical ophthalmology. Given that DMSO is inexpensive, safe, and easily formulated into therapeutic medicinal products and conventional ophthalmological drugs, this compound should be further explored and studied in the treatment of a variety of acute and chronic ocular disorders.

Cryopreservation: An Overview of Principles and Cell-Specific Considerations.

The origins of low-temperature tissue storage research date back to the late 1800s. Over half a century later, osmotic stress was revealed to be a main contributor to cell death during cryopreservation. Consequently, the addition of cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), or propylene glycol (PG), although toxic to cells at high concentrations, was identified as a necessary step to protect against rampant cell death during cryopreservation. In addition to osmotic stress, cooling and thawing rates were also shown to have significant influence on cell survival during low temperature storage. In general, successful low-temperature cell preservation consists of the addition of a CPA (commonly 10% DMSO), alone or in combination with additional permeating or non-permeating agents, cooling rates of approximately 1ºC/min, and storage in either liquid or vapor phase nitrogen. In addition to general considerations, cell-specific recommendations for hepatocytes, pancreatic islets, sperm, oocytes, and stem cells should be observed to maximize yields. For example, rapid cooling is associated with better cryopreservation outcomes for oocytes, pancreatic islets, and embryonic stem cells while slow cooling is recommended for cryopreservation of hepatocytes, hematopoietic stem cells, and mesenchymal stem cells. Yields can be further maximized by implementing additional pre-cryo steps such as: pre-incubation with glucose and anti-oxidants, alginate encapsulation, and selecting cells within an optimal age range and functional ability. Finally, viability and functional assays are critical steps in determining the quality of the cells post-thaw and improving the efficiency of the current cryopreservation methods.

Cranioplasty with Autologous Bone Flaps Cryopreserved with Dimethylsulphoxide: Does Tissue Processing Matter.

OBJECTIVE: The aim of this article was to study the outcome of patients who underwent cranioplasty with cryopreserved autologous bone after decompressive craniectomy. METHODS: Data from 74 patients were retrospectively analyzed. They were divided into groups according to the storage time and the age at cranioplasty. To assess the predictive potential for complication, factors were related to successive stages (preoperative, craniectomy, tissue processing, cranioplasty, and postoperative). Cooling and warming rates applied on bone flap were calculated. The ability to inhibit microbial growth was determined exposing bone fragments to a panel of microorganisms. The concentration of antibiotics eluted from the bone was also determined. A bone explant culture method was used to detect living cells in the thawed cranial bone. RESULTS: Hydrocephalus was significantly more frequent in pediatric patients (26.7%) than in adults (5.1%). The overall rate of bone flap resorption was 21.6% (43.7% of which required reoperation). Surgical site infection after cranioplasty was detected in 6.8% of patients. There was no correlation between infection as a postoperative complication and previous microbiological-positive culture during processing. The cause of craniectomy did not influence the risk of bone flap contamination. Vancomycin was the only antibiotic detected in the supernatant where the bone was incubated. Outgrowth from bone explants was observed in 36.8% of thawed skulls. An early start of bone flap processing at the tissue bank had a positive effect on cell viability. CONCLUSIONS: The outcome after autologous cranioplasty is a multifactorial process, which is modulated by patient-related, surgery-related, and bone-related factors.

Glycerin-preserved Human-donor Corneoscleral Patch Grafts for Glaucoma Drainage Devices.

PRECIS: Glycerin-preserved, human-donor, corneoscleral patch grafts are effective and safe for glaucoma drainage device (GDD) implantation, and they are comparable to previously reported materials. It can be preserved with the sterile technique for up to 12 months. PURPOSE: To evaluate the efficacy and safety of glycerin-preserved human donor corneoscleral tissue as the patch graft for GDD implantation. PATIENTS AND METHODS: This was a retrospective noncomparative study from the medical records of 102 eyes from 102 glaucoma patients who underwent GDD implantation by or under supervision of a single surgeon (N.K.) at the Department of Ophthalmology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand between January 2006 and December 2016. The glycerin-preserved human-donor corneoscleral tissue was used as the patch graft to cover the tube portion of GDD over the sclera. The primary outcome measure was the occurrence of patch graft-related complications. RESULTS: There were 64 males and 38 females with the mean age of 52.8±18.5 years. The underlying diseases included failed filtration surgery with primary open-angle glaucoma 32 eyes and primary angle-closure glaucoma 15 eyes, congenital glaucoma 3 eyes and secondary glaucoma 52 eyes. The mean of ocular surgeries before GDD implantation was 2.3±1.1. Patch graft-related complications included tube exposure in 4 eyes (3.9%) and wound leakage in 4 eyes (3.9%). Eyes with tube exposure underwent regrafting 3 eyes and tube reposition 1 eye. Eyes with wound leaking resolved spontaneously 2 eyes and underwent conjunctival resuturing 2 eyes. The 5-year survival rate of the corneoscleral graft was 95.7%. There was no recurrence of graft-related complications after surgical procedure to correct the complications. Postoperatively, the mean of intraocular pressure and antiglaucoma medications decreased significantly from 27.4±9.8 mm Hg and 3.8±0.93 to 13.8±6.4 mm Hg (P<0.001) and 1.6±1.5 (P<0.001) at the last visit, respectively. The mean follow-up time was 59.9 months (range, 1 to 144.7 mo). CONCLUSION: The glycerin-preserved human-donor corneoscleral tissue using as the patch graft was a safe alternative for GDD tube coverage. The patch graft-related complications was comparable to other materials.

Human sperm vitrification: A scientific report.

BACKGROUND: The sperm vitrification developed by this group is based on the ultrarapid freezing of a vitrification solution composed of a non-permeable cryoprotectant (saccharides and protein), in which previously selected spermatozoa are resuspended, free of seminal plasma, and then plunged directly into liquid nitrogen. Compared to traditional sperm freezing, vitrification does not cause chemical or physical damage to the intracellular structures and reduces the damage to the plasma membrane because no ice crystals form, thus preserving motility and DNA integrity. OBJECTIVES: This manuscript is a review of the vitrification methodology developed by the authors' research group, including studies showing the application in human reproduction therapy. MATERIALS AND METHODS: The authors perform a review of the work initiated more than a decade ago by this research group, on the implementation of sperm vitrification, a more effective technique for cryopreservation of human spermatozoa, discussing the results obtained by other authors and the projection of this technique. RESULTS AND DISCUSSION: The vitrification technique has been developed in selected spermatozoa free of seminal plasma supplemented with saccharides such as sucrose, trehalose, and dextran, together with albumin, providing a high motility rate and protective structures of the cytoskeleton. In patients, it can be used to preserve their fertility for oncological reasons, genetics, inflammatory diseases, or reproductive medicine techniques. The possibility that vitrified spermatozoa can be preserved at temperatures of -80°C can simplify sample storage, optimizing the space and time as well as operator safety. CONCLUSION: Vitrification techniques have demonstrated the preservation of selected spermatozoa without seminal plasma and with non-permeable cryoprotectants and protein. Currently, it is one of the most effective ways to maintain sperm function and has been used in in vitro fertilization or intrauterine insemination in humans, achieving healthy live births.

Soft liquid metal nanoparticles achieve reduced crystal nucleation and ultrarapid rewarming for human bone marrow stromal cell and blood vessel cryopreservation.

High warming rates during cryopreservation are crucial and essential for successful vitrification. However, realizing a faster warming rate in low-concentration cryoprotective agents appears to be challenging for conventional warming process through convective heat transfer. Herein, we developed a liquid metal (LM) nanosystem that can act as a spatial source to significantly enhance the warming rates with near-infrared laser irradiation during the warming process. The synthetic Pluronic F127-liquid metal nanoparticles (PLM NPs) displayed multiple performances with uniform particle size, superior photothermal conversion efficiency (52%), repeatable photothermal stability, and low cytotoxicity. Particularly, it is more difficult for the liquid PLM NPs with less surface free energy to form crystal nucleation than other solid NPs such as gold and Fe3O4, which is beneficial for the cooling process during cryopreservation. The viability of human bone marrow-derived mesenchymal stem cells postcryopreservation reached 78±3%, which is threefold higher than that obtained by the conventional warming method (25±6%). Additionally, the cells postcryopreservation maintained their normal attachment, proliferation, surface marker expression, and intact multilineage differentiation properties. Moreover, the results of mouse tails including blood vessel cryopreservation showed a relatively improved intact structure when using PLM NP rewarming compared with the results of conventional warming. The new LM nanosystem provides a universal platform for cryopreservation that is expected to have potential for widespread applications including bioengineering, cell-based medicine, and clinical translation. STATEMENT OF SIGNIFICANCE: In this study, we fabricated soft liquid metal nanoparticles with high photothermal conversion efficiency, repeatable photothermal stability, and low cytotoxicity. Particularly, soft liquid metal nanoparticles with less surface free energy and suppression effects of ice formation were first introduced to mediate cryopreservation. Superior ice-crystallization inhibition is achieved as a result of less crystal nucleation and ultrarapid rewarming during the freezing and warming processes of cryopreservation, respectively. Collectively, cryopreservation of human bone marrow stromal cells (HBMSCs) and mouse tails including blood vessels can be successfully performed using this new nanoplatform, showing great potential in the application of soft nanoparticles in cryopreservation.

How to improve quality of life in patients with acute leukemia and comorbid ischemic heart disease treated with anthracycline-based induction chemotherapy.

AIM: To evaluate the quality of life (QoL) parameters in patients with acute leukemia (AL) during standard induction chemotherapy, depending on the presence of concomitant ischemic heart disease and to improve them by the prevention of anthracycline cardiotoxicity with L-arginine. MATERIALS AND METHODS: A total of 147 adult AL patients (72 males and 75 females with the mean age 54.7 ± 9.3 years) were enrolled in the study. QoL assessment was performed at baseline and after induction chemotherapy using SF-36 questionnaire. Both physical and mental parameters were evaluated. RESULTS: The QoL analysis of patients with AL at the time of initial diagnosis showed extremely low QoL level in all subgroups compared with healthy individuals. It should be noted that the level of patients' QoL after achieving remission remained significantly lower than those of practically healthy, which is primarily due to the need for further long-term treatment and, probably, the fear of the disease relapse development. It was found that the administration of L-arginine during induction chemotherapy in order to reduce the risk of anthracycline cardiotoxicity development has allowed improving the QoL in patients with concomitant ischemic heart disease. CONCLUSION: L-arginine decreases the risk of anthracycline-induced myocardial injury and improves QoL in AL patients.

For whom the egg thaws: insights from an analysis of 10 years of frozen egg thaw data from two UK clinics, 2008-2017.

PURPOSE: To better understand the characteristics of patients who returned to thaw their frozen eggs to attempt conception and their outcomes. METHODS: A retrospective analysis of clinical records for all own egg thaw patients in two UK fertility clinics across 10 years, 2008-2017. RESULTS: There were 129 patients who returned to thaw their eggs, of which 46 had originally frozen their eggs for social reasons and 83 for a variety of clinical, incidental, and ethical reasons (which we have called "non-social"). Women who had frozen their eggs for social reasons were single at time of freeze, with an average age of 37.7. They kept their eggs in storage for just under 5 years, returning to use them at the average age of 42.5. 43.5% were single at time of thaw, and 47.8% used donor sperm to fertilise their eggs. Women whose eggs were frozen for non-social reasons were almost all (97.6%) in a relationship at both time of freeze and thaw. They had an average age of 37.2 at first freeze and 37.6 at thaw, having kept their eggs in storage for an average of 0.4 years. Overall, there was a 20.9% success rate among women attempting conception with frozen-thawed eggs. CONCLUSIONS: Despite widespread assumptions, many women attempting conception with thawed eggs had not initially frozen them for social reasons. Women who froze their eggs for social reasons presented distinctly and statistically different characteristics at both time of freeze and thaw to women whose eggs were frozen for non-social reasons.

Cryopreservation of Marchantia polymorpha spermatozoa.

The liverwort Marchantia polymorpha has become one of the model organisms, since it has less genetic redundancy, sexual and asexual modes of reproduction and a range of genomic and molecular genetic resources. Cryopreservation of fertile spermatozoa eliminates time, space and labor for growing and maintaining male plants in reproductive phase, and also provides an optional way to backup lines. Here we report a protocol to cryopreserve spermatozoa of M. polymorpha in liquid nitrogen. A cryoprotective solution containing sucrose, glycerol and egg yolk and controlled cooling and warming processes led to successful recovery of motile M. polymorpha spermatozoa after the cryogenic process. The survival rate and average motility of spermatozoa after cryopreservation were maintained at 71 and 54% of those before cryopreservation, respectively. Cryopreserved spermatozoa were capable of fertilization to form normal spores. The technique presented here confers more versatility to experiments using M. polymorpha and could be applied to preservation of plant spermatozoa in general.

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling.

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

Arecoline ameliorates hyperthyroid condition in mice under cold stress.

Betel nut of Areca catechu is chewed by millions of people for increased capacity to work and stress reduction, but it contains arecoline that causes hypothyroidism. The aim is to investigate the role of arecoline on thyroid activity in cold stress in mice. Arecoline treatment (10 mg/kg body wt/day, for 7 d) caused a reduction in thyroid weight and ultrastructural degeneration of thyro-follicular cells with depletion of T3 and T4 levels compared with the control mice. Cold stress (4 °C for 2 h, twice daily, for 7 d) stimulated thyroid activity ultrastructurally with an elevation of T3 and T4 levels. Arecoline treatment in cold stress suppressed thyroid activity by showing reversed changes to those of cold stress. In contrast, TSH concentrations were consistently increased under all experimental conditions. The findings suggest that cold stress causes hyperthyroidism which arecoline can ameliorate in mice.

Measuring the Densities of Aqueous Glasses at Cryogenic Temperatures.

We demonstrate a method for determining the vitreous phase cryogenic temperature densities of aqueous mixtures, and other samples that require rapid cooling, to prepare the desired cryogenic temperature phase. Microliter to picoliter size drops are cooled by projection into a liquid nitrogen-argon (N2-Ar) mixture. The cryogenic temperature phase of the drop is evaluated using a visual assay that correlates with X-ray diffraction measurements. The density of the liquid N2-Ar mixture is adjusted by adding N2 or Ar until the drop becomes neutrally buoyant. The density of this mixture and thus of the drop is determined using a test mass and Archimedes principle. With appropriate care in drop preparation, management of gas above the liquid cryogen mixture to minimize icing, and regular mixing of the cryogenic mixture to prevent density stratification and phase separation, densities accurate to <0.5% of drops as small as 50 pL can readily be determined. Measurements on aqueous cryoprotectant mixtures provide insight into cryoprotectant action, and provide quantitative data to facilitate thermal contraction matching in biological cryopreservation.

Outcome of Therapeutic Penetrating Keratoplasty Using Glycerol-Preserved Donor Corneas in Infectious Keratitis.

PURPOSE: To report the surgical outcomes and complications of therapeutic penetrating keratoplasty (TPK) using glycerol-preserved corneas in infectious keratitis. METHODS: This is a retrospective, noncomparative case series of patients with severe infectious keratitis who received TPK using glycerol-preserved corneas from 2004 to 2014 in the Department of Ophthalmology, Srinagarind Hospital, Khon Kaen University. The medical records were reviewed for baseline characteristics, visual outcomes, recurrence rate, wound integrity, secondary glaucoma, and donor cornea storage times. RESULTS: Twenty-two eyes from 22 patients were included. Age ranged from 28 to 85 years and the donor cornea sizes ranged from 7.5 to 9.5 mm. The most common causative agents were fungi (16/22, 72.7%). Eleven patients (50.0%) developed secondary glaucoma and 7 patients (31.8%) had wound leakage. Recurrence of infection was observed in 15 patients (68.2%) and 9 patients (40.9%) received enucleation or evisceration. Thirteen globes (59.1%) were saved and the final visual acuites ranged from 1/60 to light perception. The storage times of donor corneas varied between 2 days and 62 months. The length of donor cornea storage did not affect the success rate of surgical outcome. CONCLUSIONS: TPK using glycerol-preserved corneas has a high rate of secondary glaucoma and recurrence of infection with unsatisfactory visual results. These corneas may be used as temporary emergency transplants in infectious keratitis when fresh corneas are unavailable to meet demands.

New methods for cooling and storing oocytes and embryos in a clean environment of -196°C.

It is well documented that oocyte vitrification using open systems provides better results than closed systems. However, its use is limited owing to risks of contamination posed by direct exposure to liquid nitrogen and cross-contamination when stored in liquid nitrogen tanks. A device that produces clean liquid air (CLAir) having similar a temperature as liquid nitrogen and a sterile storage canister device (Esther) that keeps samples sealed in their own compartment while in regular liquid nitrogen tanks were developed. The following experiments were performed: temperature measurements, bioburden tests, vitrification and storage experiments with mice embryos and human oocytes. Results showed similar cooling rates for liquid nitrogen and liquid air. Bioburden tests of CLAir and Esther showed no contamination, while massive contamination was found in "commercial" liquid nitrogen and storage canisters. Mice blastocysts had a survival rate of over 90%, with 80% hatching rate after vitirification in CLAir and 1 week storage in Esther, similar to the fresh (control) results. Human oocytes vitrified in CLAir and in liquid nitrogen for three consecutive vitrification/warming cycles showed 100% survival, seen as re-expansion in both groups. These new systems represent a breakthrough for safe vitrification using open systems and a safe storage process generally.

Effectiveness of glucose-methanol extender for cryopreservation of Huso huso spermatozoa.

The present approach was designed to evaluate the methanol-glucose extender effects on sperm cryopreservation in beluga sturgeon, Huso huso. Sperm quality was examined by measuring post-thaw sperm motility and fertilizing rate at hatching stage. We first tested the effect of glucose concentration (0, 0.10, 0.15, 0.20 and 0.30M) in a methanol extender on post-thaw sperm motility. The optimal cryopreservation conditions were found to be 0.2M glucose in the extender. Then, motility and fertilization rates of sperm cryopreserved with 0.2M glucose and 10% methanol (GM) were compared to Tris-sucrose-KCl in 10% methanol extender (TSKM). Additionally, sperm motility and fertilizing ability in relation to 15 and 30min equilibration in GM extender before and after cryopreservation were measured. Higher post-thaw sperm motility duration and percentage as well as fertilization rate were obtained with the GM extender when compared to TSKM extender. Equilibration of sperm in extender did not affect the motility quality of either fresh-diluted or frozen/thawed sperm, while fertilization rate showed a significant decline alone after 30min of post-thaw storage. Our results indicated that the use of a simple extender consisting of 0.2M glucose in 10% methanol can be an alternative cryopreservation method to those previously described for sturgeons.

Liposome-based semen extender is suitable alternative to egg yolk-based extender for cryopreservation of buffalo (Bubalus bubalis) semen.

Demand for alternative of egg yolk in freezing extenders have increased in recent years due to variability in egg yolk composition, risk of microbial contamination and presence of steroid hormones. The alternative to egg yolk-based extender (EY) can be soya lecithin-based extender (SL) and liposome-based extender (LP). However, the efficacy of SL is still a matter of debate. Few studies have been performed on the effect of LP but to date evaluation of buffalo semen cryopreserved in LP has not been studied. Therefore, this study was designed to compare SL and LP with conventional EY for evaluation of post-thaw quality of buffalo semen. Results showed that total, progressive and rapid sperm motility were found significantly higher (P<0.05) in LP among these extenders. In vitro assessment of post-thaw sperm longevity has also resulted in better maintenance of sperm kinetics and motility in LP in comparison to other extenders. Furthermore, sperm cryopreserved in LP travelled significantly more (P<0.05) distance in cervical mucus as compared to SL and EY. Therefore, it can be concluded that the LP is more efficient than SL and EY for the cryopreservation of buffalo semen.

Human embryo cryopreservation-methods, timing, and other considerations for optimizing an embryo cryopreservation program.

The contribution of embryo cryopreservation to the birth rate per in vitro fertilization cycle has escalated from a rare subsidy to a vital tool that is called upon to augment the cycle outcome. Embryology laboratories must identify the embryo stage, quality criteria and methodology that will optimize their ability to preserve each embryo's reproductive potential. This chapter reviews the principles of cryopreservation, outcomes based on embryo stage and cryopreservation method and benchmarks that may be employed by the laboratory to measure the performance of their embryo cryopreservation program.