A simple and economic protocol for efficient in vitro fertilization using cryopreserved mouse sperm.

The advent of genome editing tools like CRISPR/Cas has substantially increased the number of genetically engineered mouse models in recent years. In support of refinement and reduction, sperm cryopreservation is advantageous compared to embryo freezing for archiving and distribution of such mouse models. The in vitro fertilization using cryopreserved sperm from the most widely used C57BL/6 strain has become highly efficient in recent years due to several improvements of the procedure. However, purchase of the necessary media for routine application of the current protocol poses a constant burden on budgetary constraints. In-house media preparation, instead, is complex and requires quality control of each batch. Here, we describe a cost-effective and easily adaptable approach for in vitro fertilization using cryopreserved C57BL/6 sperm. This is mainly achieved by modification of an affordable commercial fertilization medium and a step-by-step description of all other necessary reagents. Large-scale comparison of fertilization rates from independent lines of genetically engineered C57BL/6 mice upon cryopreservation and in vitro fertilization with our approach demonstrated equal or significantly superior fertilization rates to current protocols. Our novel SEcuRe (Simple Economical set-up for Rederivation) method provides an affordable, easily adaptable and harmonized protocol for highly efficient rederivation using cryopreserved C57BL/6 sperm for a broad application of colony management in the sense of the 3Rs.

Sperm membrane integrity and stability after selection of cryopreserved ovine semen on colloidal solutions.

The aim of this study was to evaluate the effect of four methods of sperm selection, on the integrity and stability of the plasma membrane, integrity of the acrosomal membrane and spermatic morphology in frozen/thawed ovine semen. Two types of colloidal silica: colloidal silica-silane and colloidal silica-polyvinylpyrrolidone (PVP), and two aliquots: 1 and 4 ml, were used for sperm selection. Probes FITC-PSA and PI were used to measure the integrity of the plasma and acrosomal membranes. Plasma membrane stability was measured, using fluorescent probes M540 and YOPRO1. Effective reduction in the incidence of spermatozoa with acrosomal pathologies was only achieved using 1 ml colloidal silica-silane. All methods were efficient in select viable and unreacted spermatozoa. Only methods using 1 ml of silica were efficient in decrease spermatozoa stained by PI (death). Methods using silica colloidal-silane were more efficient to decrease apoptotic cells after selection when compared to silica colloidal-PVP. In conclusion, sperm selection in colloidal silica-silane and colloidal silica-PVP improved sperm quality when compared to the controls. The method using 1 ml of colloidal silica-silane is the preferred method because its effectiveness and lower cost.

Applications and cost benefits of sexed semen in pasture-based dairy production systems.

Sexed semen technology is now commercially available in many countries around the world, and is primarily used in dairy cattle breeding. Sperm are sorted by flow cytometry on the basis of a 4% difference in DNA content between sperm containing X and Y chromosomes. Despite reliably producing a 90% gender bias, the fertility of the sexed semen product is compromised compared with conventional semen. The negative implications of the reduced fertility of sexed semen are amplified in seasonal systems of dairy production, as the importance of fertility is greater in these systems compared with year-round calving systems. A review of the literature indicates that conception rates (CR) to 1st service with frozen-thawed sexed semen are ~75% to 80% of those achieved with conventional frozen-thawed semen. Preliminary results from a large-scale field trial carried out in Ireland in 2013 suggest that significant improvements in the performance of sexed semen have been made, with CR of 87% of those achieved with conventional semen. The improved fertility of a sexed semen product that delivers a 90% gender bias has considerable implications for the future of breeding management in pasture-based dairy production systems. Sexed semen may facilitate faster, more profitable dairy herd expansion by increasing the number of dairy heifer replacements born. Biosecurity can be improved by maintaining a closed herd during the period of herd expansion. In a non-expansion scenario, sexed semen may be used to increase the value of beef output from the dairy herd. The replacement heifer requirements for a herd could be met by using sexed semen in the 1st 3 weeks of the breeding season, with the remaining animals bred to beef sires, increasing the sale value over that of a dairy bull calf. Alternatively, very short gestation sires could be used to shorten the calving interval. Market prices have a considerable effect on the economics of sexed semen use, and widespread use of sexed semen should be restricted to well managed herds that already achieve acceptable herd fertility performance.

Long-term preservation of freeze-dried mouse spermatozoa.

Many genetically engineered mice strains have been generated worldwide and sperm preservation is a valuable method for storing these strains as genetic resources. Freeze-drying is a useful sperm preservation method because it requires neither liquid nitrogen nor dry ice for preservation and transportation. We report here successful long-term preservation at 4 °C of mouse spermatozoa freeze-dried using a simple buffer solution (10mM Tris, 1mM EDTA, pH 8.0). Offspring with fertility were obtained from oocytes fertilized with freeze-dried spermatozoa from C57BL/6 and B6D2F1 mouse strains stored at 4 °C for 3 years. This freeze-drying method is a safe and economical tool for the biobanking of valuable mouse strains.

Comparative costs of programmes to conserve chicken genetic variation based on maintaining living populations or storing cryopreserved material.

1. There have been substantial losses of chicken lines kept for research in recent years and the objective of this research was to critically review alternative methods of preserving genetic resources. 2. The costs of programmes using living populations, semen cryopreservation and reconstitution, and ovary and semen cryopreservation and reconstitution were evaluated over 20 years using biological parameters of cryopreservation and population reconstitution that were derived from the literature. 3. Keeping live populations was most cost effective for periods of up to three years, but keeping live populations is increasingly difficult to justify with longer periods and any research population that will not be used within five years should be cryoconserved and in situ maintenance discontinued. 4. The rapid reconstitution possible using ovaries and semen would allow the inclusion of cryopreserved material in a short-term research project with the cost of recovery included in the budget. The low cost of cryoconservation suggests that all avian material should be conserved and reconstituted when needed for research.

The current value of frozen-thawed boar semen for commercial companies.

Opportunities for use of frozen-thawed semen (FTS) must address genetic advancement and fertility for developing practical models for use. Concerns about slowed genetic gains, lowered fertility and additional costs may limit use of FTS. However, FTS is presently used for international exchange among nucleus farms to help maintain genetic diversity. Use of FTS beyond several days can provide increased flexibility for on-farm use and allow additional time for disease tests. There may be potential for use of FTS for short-term banking during periods of low demand and while sires await breeding value tests. Opportunities exist for FTS use in long-term banking of sire lines, creation of semen pools for genetic progress evaluation and for use in emergencies. Advancing FTS technology will require the following: (i) more efficient production of doses; (ii) improved fertility with single sire or pooled matings; (iii) education and training; and (iv) models for FTS use and economics for use under various scenarios.

Colloid centrifugation of boar semen.

Colloid centrifugation of boar semen has been reported sporadically for at least the last two decades, beginning with density gradient centrifugation (DGC) and progressing more recently to single layer centrifugation (SLC). Single layer centrifugation through a species-specific colloid has been shown to be effective in selecting the best spermatozoa (spermatozoa with good motility and normal morphology) from boar sperm samples. The method is easier to use and less time-consuming than DGC and has been scaled-up to allow whole ejaculates from other species, e.g. stallions, to be processed in a practical manner. The SLC technique is described, and various scale-up versions are presented. The potential applications for SLC in boar semen preservation are as follows: to improve sperm quality in artificial insemination (AI) doses for 'problem' boars; to increase the shelf-life of normal stored sperm samples, either by processing the fresh semen before preparing AI doses or by processing the stored semen dose to extract the best spermatozoa; to remove pathogens (viruses, bacteria), thus improving biosecurity of semen doses and potentially reducing the use of antibiotics; to improve cryosurvival by removing dead and dying spermatozoa prior to cryopreservation; to select spermatozoa for in vitro fertilization. These applications are discussed and practical examples are provided. Finally, a few thoughts about the economic value of the technique to the boar semen industry are presented.

Cost-effectiveness of vasectomy reversal.

In this era of cost-consciousness and containment, it is imperative to examine not only treatment outcomes but also cost of these treatments. With improvements of in vitro fertilization outcome and continued development of less-invasive sperm retrieval methods, physicians and couples must examine all options available after surgical sterilization. Vasectomy reversal remains the gold standard of treatment; however, certain situations may be present in which sperm acquisition/in vitro fertilization may be a better option. A physician's responsibility is to present all options with the pros and cons of each, including cost, to help arrive at an informed decision.

A simple, inexpensive and successful freezing method for curimba Prochilodus lineatus (Characiformes) semen.

The cryopreservation of fish sperm provides a tool by which reproduction is optimized and thereby larval production is increased. The aims of this study were to evaluate the effects of cryosolutions, motility-activation media, straw volumes and thawing temperatures on the post-thaw motility of curimba semen. Furthermore, semen cryopreserved in a simple and inexpensive cryosolution and that yielded excellent post-thaw motility was tested for fertility. Semen was diluted in each of the eight cryosolutions in a factorial of two cryoprotectants (DMSO and methylglycol) x four extenders (0.9% NaCl, 5% glucose, BTS and M III). Diluted semen was frozen in 0.5-mL straws in a nitrogen vapor vessel. Sperm motility was evaluated after thawing (60 degrees C water bath for 8s) and activation with a total of four different activation media (distilled water, 0.15% NaCl, 0.29% NaCl or 1% NaHCO(3)). To evaluate straw volume and thawing temperature, semen was diluted in 5% glucose and methylglycol and frozen in 0.5- and 4.0-mL straws. Half of the 0.5-mL straws were thawed in a water bath at 60 degrees C for 8s and the other half at 30 degrees C for 16s. The 4.0-mL straws were thawed at 60 degrees C for 24s only. In the last experiment, semen cryopreserved in 5% glucose and methylglycol, 0.5-mL straws, and thawed at 60 degrees C for 8s was tested for fertility. The results of these comparisons are presented and show that curimba semen can be successfully cryopreserved in a simple glucose solution combined with methylglycol as cryoprotectant, in 0.5-mL straws, yielding motility rates between 86% and 95% and fertilization rates between 47% and 83%.

Efficiency of short-term storage of equine semen in a simple-design cooling system.

Five experiments tested the efficiency of a simple, low-cost system (CP) for cooling and storing equine semen at 2.0 degrees C for 24 h and 48 h. Pantaneiro stallions of known fertility were used. Semen quality was evaluated for progressive motility (PM), plasma membrane integrity (PMI), and pregnancy rate. Experiment 1 showed that PM and PMI were similar between CP and the control (Equitainer) in cooled semen. In Experiment 2, the influence was evaluated of combinations (four treatments) of two volumes (50/100 ml) and two sperm concentrations (500/750x10(6)) on sperm quality of semen cooled and preserved by CP (cooling system replaced at 24 h). While PM decreased gradually from before cooling to 24 h and 48 h, PMI decreased only at the least and greatest sperm volume and concentrations. Storage time did not affect PMI. Results from Experiment 3 showed that CP maintained semen PM>or=30% in all samples 24 h after cooling and decreased to about 70% 42 h after cooling. Results from Experiments 4 and 5 confirmed semen quality after cooling and storage (24 h and 48 h, respectively), achieving a 69% pregnancy rate in the first estrous cycle when insemination occurred. Thus, the CP system is satisfactory for cooling and preserving equine semen for up to 48 h.

Sperm harvesting and cryopreservation during vasectomy reversal is not cost effective.

OBJECTIVE: To determine whether sperm harvesting and cryopreservation at the time of vasectomy reversal is cost-effective. DESIGN: Model of actual costs and results at five institutions. SETTING: Multicenter study comprising five centers, including university hospitals and private practices. PATIENT(S): Men undergoing vasectomy reversal. INTERVENTION(S): We established two models for vasectomy reversal. The first model was sperm harvesting and cryopreservation at the time of vasectomy reversal. The second model was sperm harvesting at the time of IVF only if the patient remained azoospermic after vasectomy reversal. Vasectomy reversal procedures modeled included bilateral vasovasostomy and bilateral epididymovasostomy. The costs for each procedure at the five institutions were collated and median costs determined. MAIN OUTCOME MEASURE(S): Median cost of procedure and calculated financial comparisons. RESULT(S): The median cost of testicular sperm extraction/cryopreservation performed at the time of bilateral vasovasostomy was $1,765 (range, $1,025-$2,800). The median cost of microsurgical epididymal sperm aspiration or testicular sperm extraction with cryopreservation performed at the time of epididymovasostomy was $1,209 (range, $905-$2,488). The average of the median costs for percutaneous sperm aspiration or testicular sperm aspiration for those patients with a failed vasectomy reversal was $725 (range, $400-$1,455). CONCLUSION(S): Sperm retrieval with cryopreservation at the time of vasectomy reversal is not a cost-effective management strategy.

Use of frozen semen to avoid human immunodeficiency virus type 1 transmission by donor insemination: a cost-effectiveness analysis.

OBJECTIVE: Reappraisal of current guidelines mandating frozen-thawed semen. DESIGN: Cost-effectiveness analysis comparing the use of frozen semen with the use of fresh semen from the same donors without a second antibody test. SETTING: A Markov model computer simulation. PATIENT(S): A theoretical cohort of 80000 women whose husbands are azoospermic. INTERVENTION(S): Simulation with calculation of costs and payoffs. MAIN OUTCOME MEASURE(S): Total lifetime direct health care costs, costs per live birth, life expectancy, quality adjusted life years (QALY), marginal cost effectiveness (dollar/QALY). RESULT(S): If all 80000 women who undergo donor insemination in the United States each year chose to use fresh semen from donors screened according to the current practice guidelines but without semen cryopreservation, there would be 8881 more births and the mean cost per live birth would be US dollars 15501 less. One recipient would become infected with HIV-1 every 5.1 years, during which time over 180000 noninfected children would be born. The average life expectancy of recipients would be reduced by 2 days, but their quality-adjusted life expectancy would increase by over 1 month. Medicolegal costs to physicians would need to exceed US dollars 780 million per infection to equalize the cost effectiveness of the fresh and frozen policies. CONCLUSION(S): The guidelines should be revised to allow the use of fresh semen by informed recipients.

Semen cryopreservation of small abalone (Haliotis diversicolor supertexa).

Methods for cryopreserving spermatozoa and maximizing fertilization rate in Taiwan small abalone, Haliotis diversicolor supertexa, were developed. The gametes (spermatozoa and eggs) of small abalone were viable 3 h post-spawning, with fertilization, and development rate decreasing with time. A minimum of 10(2) cell/ml sperm concentration and a contact time of 2 min between gametes is recommended for artificial insemination of small abalone eggs. Eight cryoprotectants, dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), ethylene glycol (EG), propylene glycol (PG), butylene glycol (BG), polyethylene glycol, glycerol and methanol, were tested at concentrations between 5 and 25% to evaluate their effect on motility of spermatozoa exposed to cryoprotectant for up to 60 min at 25 degrees C before freezing. The least toxic cryoprotectant, 10% DMSO, was added to artificial seawater (ASW) to formulate the extender for freezing. Semen was diluted 1:1 with the extender, inserted into 1.5 ml microtubes and frozen using a cooling rate between -3.5 and -20 degrees C/min to various transition temperatures (0, -30, -60, -90 and -120 degrees C), followed by transfer and storage in liquid nitrogen (-196 degrees C). The microtubes were thawed from +45 to +145 degrees C/min. Spermatozoa, cooled to -90 degrees C at a cooling rate of -12 or -15 degrees C/min and then immersed in liquid nitrogen, had the best post-thaw motility. Post-thaw sperm motility was markedly reduced compared to fresh sperm. More frozen-thawed spermatozoa are required to achieve fertilization rates comparable to those achieved using fresh spermatozoa.

Is a review of standard procedures for cryopreservation needed?: safe and effective cryopreservation-should sperm banks and fertility centres move toward storage in nitrogen vapour?

A recent consultation documentation by the Human Fertilisation and Embryology Authority (HFEA) which focused on the safe cryopreservation of gametes and embryos highlighted the need for a review of the way that fertility clinics in the UK store potentially infective material. The main points for consideration were to: (i) ensure containers used for cryopreservation are guaranteed by manufacturers to withstand low temperatures; (ii) use secondary containers, i.e. 'double bagging' of samples if stored in the liquid phase; and (iii) store in nitrogen vapour as a 'safer' alternative. In this article we examine a number of issues related to vapour storage which need careful consideration, including safety, cost and the effectiveness of various storage techniques in maintaining gamete and embryo viability. We also discuss the effectiveness of vapour storage in comparison with current liquid nitrogen storage techniques. In conclusion, we propose that fertility clinics should be compelled to review their cryopreservation procedures, not just because of new legislation or indeed fear of litigation but by a moral obligation.

Reproductive issues for men with cancer: technology and nursing management.

PURPOSE/OBJECTIVES: To review the current medical technologies available to protect the reproductive potential of adult males undergoing sterilizing cancer treatments; to describe the attributes and limitations of these technologies and how oncology nurses can access them for the patient; and to discuss psychosocial elements, including the legal considerations of oncology nurses who counsel patients. DATA SOURCES: Quantitative data from personal clinical records; personal clinical experience; published articles, abstracts, and books identified by bibliographic data base searches; and consultation with lawyers. DATA SYNTHESIS: Cancer treatment can have severe and adverse long-term iatrogenic effects on male fertility. Medical technologies that protect male reproduction potential from sterilizing procedures have progressed from unreliable to clinically practical over a period of 20 years. The present clinical means for preserving the potential reproductive capacity of men at risk is cryopreservation of sperm before treatment begins, followed by assisted reproductive technology when pregnancy is desired. Medical, legal, and religious issues relevant to counseling are involved. CONCLUSIONS: Current reproductive technology provides realistic hope for future procreation by men facing sterilizing cancer treatment. IMPLICATIONS FOR NURSING PRACTICE: Nursing intervention primarily involves providing patient counseling and arranging patient access to cryopreservation facilities. Oncology nurses can assist men making these types of reproductive decisions by assessing their medical and psychological need for information and by counseling them in regard to human sexuality, the fertility risk of oncologic therapy, the availability of reproductive interventions, and the social ramifications of using stored semen.

Isolamento do vírus da rinotraqueíte infecciosa bovina do sêmen congelado para comercializaçäo / Isolation of infectious bovine rhinotracheitis virus from semen samples of a Brazilian AI study

Bovine herpesvirus 1 (HVB1) was isolated in vitro using two MDBK cells lines from Brazilian commercial semen. The identification of the virus was carried out by direct fluorescent antibody test in cultures showing cytophatic effect

Preservation techniques for human semen.

Sperm banking with the relatively simple and economical biologic freezer (Linde BF-5; Union Carbide Corporation, Linde Division, New York, N.Y.) was compared with controlled-rate freezer (Linde CRF-1; Union Carbide), using different freezing rates and cryoprotective media. The results were assessed by comparing prefreeze and post-thaw sperm motility. Our results demonstrate a significant loss of sperm motility with both techniques; however (1) complex cryoprotective medium (CPM) containing egg yolk produced better results than did 10% glycerol, (2) the rate of freezing at 10 degrees C versus 1 degrees C/minute did not produce a significant difference, and (3) results with the controlled-rate freezer and BF-5 were similar. We therefore conclude that sperm banking can be accomplished in a relatively simple and reliable way by using the BF-5 and CPM.

Practical aspects of banking patient's semen for future artificial insemination.

The necessary information is provided for the urologist to answer the question, "Doctor, can I freeze my semen before surgery and use it later to impregnate my wife?" A listing of all semen banks presently storing homologous semen and registered with the American Association of Tissue Banks is presented. The methods used, ancillary tests that are done or recommended, and costs are provided. The logistics of arranging for deposits and withdrawals are also discussed.

Analysis of the feasibility of storing human semen for future artificial insemination.

Patients about to undergo vasectomy, retroperitoneal lymph node dissection, pelvic radiotherapy or chemotherapy frequently wish to store semen for future artificial insemination. The probability that this will result in a pregnancy is approximately 33% and the logistics are complicated. These problems are discussed and a cost analysis is presented. A list of semen banks presently storing homologous human semen and registered with the American Association of Tissue Banks also is presented.