286 PRODUCTION OF NORMAL MICE USING LONG-TERM PRESERVED MOUSE SPERMATOZOA WITHOUT FREEZING

Preservation of mammalian spermatozoa now plays an important role in fertility treatment, in generating hybrid animals, and in protecting endangered or extinct species. To date, the most common method of sperm preservation is freezing in liquid nitrogen (LN(2)). However, this method requires constant supplementation of the LN(2) and also involves some safety issues in transporting LN(2). Here we describe a new sperm preservation method that does not involve freezing. Mouse spermatozoa were cultured in four basic media (HEPES-CZB, potassium simplex optimization medium with amino acids [KSOMaa], K(+)-rich nuclear isolation medium [NIM], and PBS) with or without 10% bovine serum albumin (BSA) or 15% Ficoll as a protectant, and preserved in a refrigerator for up to 6 mo. These preserved sperm were then injected into fresh oocytes and cultured to the blastocyst stage in vitro or transferred into recipient females to demonstrate their genetic integrity. The results of sperm preservation for 1 mo suggested that NIM and PBS were better media than HEPES-CZB or KSOMaa and that BSA and Ficoll could improve either blastocyst or full-term development. Surprisingly, 18 pups were obtained using spermatozoa stored in these media for 6 mo. Moreover, this new method allowed easy production of healthy offspring even after transport of spermatozoa between two countries by aircraft at room temperature. In conclusion, this method allows for easy long-term preservation of mouse spermatozoa in a simple, modified medium at refrigerator temperature with very low cost and wide application.

Sperm preservation is an important technique for maintaining valuable genetic resources in biomedical research and wildlife. In the mouse, the sperm cryopreservation method has been established and adopted by large-scale sperm preservation projects in cryobanks. Recently, a new sperm preservation method using freeze-drying has been studied in various mammals. Freeze-drying is the ultimate method by which sperm can be preserved long term in a refrigerator (4 °C). And it is possible to realize easy and safe transportation of sperm at an ambient temperature that requires neither liquid nitrogen nor dry ice. Furthermore, it has been demonstrated that the fertilizing ability of sperm cryopreserved or freeze-dried by the methods described in this chapter is well maintained during long-term preservation. This chapter introduces the latest protocols for cryopreservation and freeze-drying of mouse sperm, and the anticipated results of the fertilizing ability of these sperm preserved long-term.

Long-term preservation of freeze-dried mouse spermatozoa

The generally accepted method for sperm preservation is by freezing in liquid nitrogen at -196 EC. However, this requires a constant replacement of liquid nitrogen for long-term preservation. Development of preservation techniques for male gametes at room temperature would allow us to store them with a simple and cost-effective manner. It has been known that food can be preserved in salt for long periods at room temperature. Therefore, in this study, we have tried to develop a new sperm preservation method at room temperature using a powder of salt and sugars. Whole cauda epididymides of mature B6D2F1 mouse were placed directly in a powder of pure salt (NaCl), sugars (glucose, raffinose or trehalose) or without any additives (untreated control) for 1 day to 1 month, at room temperature. Epididymides were then rehydrated with Hepes-CZB or NIM medium for 15 min at 4EC, and the sperm were collected by disrupting the epididymis using tweezers. In some experiments, cauda epididymides were gradually dehydrated using different concentrations of sugars in water, to avoid the rapid water replacement damage, and rehydration time was extended to avoid insufficient rehydration. The quality of stored spermatozoa was evaluated by their oocyte activation capacity and developmental potential after intracytoplasmic sperm injection (ICSI). When the cauda epididymis was stored for 1 week, there were no significant differences in the oocyte activation capacity of spermatozoa when compared to the fresh control, irrespective of storage conditions (stored: n= 533, fresh: n= 328; 88 to 100% vs 98%). However, when the cauda epididymis was stored for 1 month, the rate of activated oocytes decreased significantly in the untreated control (n= 130; 5%), whereas the cauda epididymis stored in salt or sugar showed that most spermatozoa still retained oocyte activation capacity after ICSI (n= 251; 83 to 95%). Activated oocytes formed male and female pronuclei which appeared normal and extrudes a second polar body 6 h after ICSI. We also examined pronuclear morphology and methylation status of histone H3 lysine 9 (H3K9) in zygotes by immunofluorescence staining, but we did not observe any differences between zygotes fertilized with stored and fresh spermatozoa (stored: n= 71, fresh: n= 30). However, the developmental ability of zygotes generated by ICSI using stored sperm showed a significant decrease. When epididymides were stored in glucose or raffinose for 1 day at room temperature, only 2% and 17% of embryos developed to blastocyst (glucose: n= 203, raffinose: n= 132), and after transfer into recipient females, only 1% and 7 % of them developed to full term (glucose: n= 208, raffinose: n= 182), respectively. However, when epididymides were stored for 1 month in glucose or raffinose, or stored in salt or trehalose for 1 day, none of embryos developed to the blastocyst stage. This could not be improved even when the epididymis was dehydrated gradually (n= 162) and rehydration time was extended (n= 259). We examined the cause of developmental arrest in embryos injected with sperm stored for 1 day and found that 100% (NaCl) or 87.8% (glucose) embryos showed aberrant chromosome segregation at the 2-cell stage (fresh: 7%). In conclusion, although the activation factor(s) is maintained in sperm preserved with salt or sugar for at least 1 month at room temperature, their developmental capacity were lost due to the abnormal distribution of the chromosomes.

Effects of two vitrification protocols on the developmental potential of human mature oocytes

Cryopreservation is one of the commonly methods used in spermatozoa preservation in which sperm is frozen and stored in the container of liquid nitrogen. The frozen sperm is still motile after thawing, so it is possible to use it in both artificial insemination and in vitro fertilization to produce an embryo. However, this technique needs a continuous supply of liquid nitrogen and a container as a place to store the frozen sperm. The advanced technique in microinjection has led the possibility of using immotile sperm to fertilize oocyte. Therefore, the sperm preservation method may be simplified because the motility of sperm has not been taken into consideration in fertilization compared to the previous method. Freeze-drying sperm is the proposed method in which the sperm is frozen and sublimated using freeze-drying machine to produce freeze-dried sperm. The freeze-dried sperm might be stored in room temperature or in refrigerator. Several reports have claimed that freeze-dried sperm is not motile but it still has capability to fertilize oocyte, even produces offspring, because its DNA remains intact. Key words: Cryopreservation, freeze-drying, sperm, oocyte

Fertilization and embryo development in fresh vs. vitrified oocytes with sperm from men with nonobstructive azoospermia.

Molecular profiling of human oocytes after vitrification strongly suggests that they are biologically comparable with freshly isolated gametes

Which reproductive treatment outcomes are observed in women who underwent elective oocyte cryopreservation (EOC) and who returned to the clinic with a desire for a child? Whether to warm oocytes or to first use fresh own oocytes for ART depends on age upon returning, but both strategies result in favorable reproductive outcomes. Most affluent countries have observed a trend toward postponement of childbearing, and EOC is increasingly used based on the assumption that oocytes cryopreserved at a younger age may extend a woman's reproductive lifespan and mitigate her age-related fertility decline. Although most follow-up studies after EOC have focused on women who requested oocyte warming, a substantial proportion of women who do not conceive naturally will embark on fertility treatment without using their cryopreserved oocytes. Reports on reproductive outcomes in past EOC users are scarce, and the lack of reproductive treatment algorithms in this group of women hampers counseling toward the most efficient clinical strategy. This retrospective observational single-center study encompasses 843 women who had elective oocyte vitrification between 2009 and 2019 at our fertility clinic. Women who underwent fertility preservation for medical or oncological reasons were excluded. This study describes the outcomes of the diverse reproductive treatment strategies performed until May 2022 in women returning to our clinic to attempt motherhood. Using descriptive statistics, patient characteristics and data of ovarian stimulation (OS) of EOC cycles were analyzed, as well as data related to OS and laboratory data of ART in women who pursued fertility treatment with and/or without using their cryopreserved oocytes. The primary outcome was live birth rate (LBR) per patient after oocyte warming and after ART using fresh oocytes. Secondary outcomes were return rate, utilization rate of the cryopreserved oocytes, laboratory outcomes upon return, and LBR per embryo transfer. A multivariable regression model was developed to identify factors associated with the decision to thaw oocytes as the primary strategy and factors associated with ongoing pregnancy upon return to the clinic. A total of 1353 EOC cycles (mean ± SD, 1.6 ± 0.9 per patient) were performed. At the time of EOC, the mean age was 36.5 ± 2.8 years, mean anti-Müllerian hormone (AMH) was 2.3 ± 2.0 ng/ml, and 174 (20.6%) women had a partner. On average, 13.9 ± 9.2 mature oocytes were cryopreserved. Two hundred thirty-one (27.4%) women returned to the clinic, an average of 39.9 ± 23.4 months after EOC. Upon returning, their mean age was 40.4 ± 3.1 years, mean AMH was 1.5 ± 1.5 ng/ml, and 158/231 (68.3%) patients had a partner. As a primary approach, 110/231 (47.6%) past EOC users embarked on oocyte warming, 50/231 (21.6%) had intrauterine insemination, and 71/231 (30.7%) had ART using fresh own oocytes. Cumulative LBR (CLBR) was 45.9% (106/231) notwithstanding a miscarriage rate (MR) of 30.7% (51/166) in the entire cohort. In total, 141 women performed oocyte warming at some stage in their treatment trajectory. A subset of 90/231 (39.0%) patients exclusively had oocyte warming (41.6 ± 3.0 years, with 10.0 ± 5.2 oocytes warmed per patient). 52/231 (22.5%) patients exclusively had ART using fresh own oocytes (mean age of 39.0 ± 2.8 years, with 9.9 ± 7.4 mature oocytes retrieved per patient). CLBR was 37/90 (41.1%) in the oocyte warming-only group and 25/52 (48.1%) in the OS-only group. MR/transfer was 25.0% and 29.3% in the oocyte warming-only group and the OS-only group, respectively. Both sample size and the retrospective design are limitations of this study. The decision to embark on a specific reproductive treatment strategy was based on patient preference, after counseling on their treatment options. This precludes direct comparison of the efficiency of reproductive treatment options in past EOC users in this study. Reporting on clinical outcomes of women who underwent EOC and returned to the clinic to embark on divergent reproductive treatment strategies is mandatory to establish guidelines for best clinical practice in this growing patient population. None. N/A.

Many wild equids are at present endangered in the wild. Concurrently, increased mechanization has pushed back the numbers of some old native horse breeds to levels that are no longer compatible with survival of the breed. Strong concerns arose in the last decade to preserve animal biodiversity, including that of rare horse breeds. Genome Resource Banking refers to the cryostorage of genetic material and is an approach for ex situ conservation, which should be applied in combination with in situ conservation programmes. In this review, we propose that, owing to the great reproductive similarity among the different members of the genus Equus, the domestic horse can be used to optimize cryopreservation and embryo production protocols for future application in wild equids. We will give this hypothesis a scientific underpinning by listing successful applications of epididymal sperm freezing, embryo freezing, intracytoplasmic sperm injection, oocyte vitrification and somatic cell nuclear transfer in domestic horses. Some ART fertilization methods may be performed with semen of very low quality or with oocytes obtained after the death of the mare.

Gamete preservation techniques are essential in animal husbandry as well as in assisted reproduction for humans. In this research we attempted to use 3 different sperm preservation techniques in combination with newly developed techniques for intracytoplasmic sperm injection (ICSI) to fertilize eggs of a teleost fish, the Nile tilapia (Oreochromis niloticus). Of 47 eggs injected with fresh sperm, 11 (23%) were fertilized, 5 developed abnormally, and 4 developed normally and hatched; from these, one grew to adulthood. Nuclear DNA content of 4 of the abnormal embryos indicated that they were diploid. Flow cytometric analysis of a blood sample from the surviving ICSI fish collected 2 months after fertilization indicated that the fish was diploid. Of 45 eggs injected with cryopreserved sperm, 9 (20%) developed to the blastula stage. Of 40 eggs injected with sperm preserved in 70% methanol, none were fertilized. No injections were possible with freeze-dried Nile tilapia sperm owing to technical difficulties during manipulation. Although the findings described here are limited, they provide the first steps toward using sperm preservation methods in addition to cryopreservation for fertilization in fishes.

BACKGROUND:Mouse sperm can be stored for long or short-time periods. Nevertheless long-term storage leds to significantly reduced sperm quality and fertility because of cryodamage. Thus, in the storage of semen in mice, it is necessary to focus on media and temperatures that gives good results in short-term storage. OBJECTIVE:To determine favorable media for short-term storage of mice spermatozoa by evaluating progressive motility, viability, membrane function integrity, acrosome integrity and fragmented DNA rates at various storage temperatures . MATERIALS AND METHODS: Mouse spermatozoa were collected from epididymides of mature CD1 males and s amples were stored at 24°C and 4°C for 60 h.RESULTS: Motility, viability and membrane function of mice spermatozoa were greatest when stored in KSOM media. Motility and viability were not different when stored at refrigerator or room temperature in KSOM compared to HTF or PBS mediums for 48 h, but were after 60 h . There was n't any significant variation in terms of acrosome integrity in different preservation conditions. Fragmented DNA rates were similar in fresh sperm with KSOM and HTF media, while there was higher damage in PBS medium at 60 h . Overall, sperm parameters were affected significantly by the time of storage and type of preservation medium, and PBS extender was not suitable for mice spermatozoa at room and refrigerated temperatures as it caused the lowest progressive motility, viability, membrane function integrity and the highest DNA damage . CONCLUSION: Mice spermatozoa stored in KSOM retained the best sperm quality parameters both 24°C and 4°C for the first 48 h.

PGT in fresh and frozen donor oocyte cycles

Development of feline embryos produced using freeze-dried sperm

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at 4 degrees C. Samples frozen without cryoprotection were maintained at -196 degrees C. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%, respectively; P > 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P > 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P < 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P < 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P > 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 1.5 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 2) long-term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple, and efficient but, like all routine sperm storage methods, requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying, but sperm can then be stored at 4 degrees C and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.