Cryodevices developed for minimum volume cooling vitrification of bovine oocytes

Abstract Unfertilized bovine oocytes can be efficiently cryopreserved only when an extremely rapid cooling rate (>20,000°C/min) is applied to oocytes with a very limited amount of surrounding vitrification solution. This protocol is defined as minimum volume cooling (MVC) vitrification. Various types of cryodevices, such as open pulled straw, Cryoloop, and Cryotop, have been developed to accelerate the cooling efficacy. Furthermore, hollow fibers with nano‐scale pores, triangle nylon mesh sheets, and multilayer silk fibroin sheets have been optimized for the loading of large quantities of oocytes and/or the subsequent removal of excess vitrification solution, without requiring skillful operation to transfer individual oocytes using fine capillaries. This article provides an up‐to‐date review of cryodevices suitable for the MVC vitrification of bovine oocytes at the immature (germinal vesicle‐) and mature (metaphase II‐) stages.


| INTRODUCTION
Meiosis-arrested bovine oocytes are very large spherical cells (diameter = 140 μm, enclosed with an acellular zona pellucida) surrounded by cumulus cell layers after ovulation and prior to sperm penetration. Cryopreserved oocytes provide a valuable resource for embryo production via assisted reproductive technology (ART), including in vitro fertilization (IVF), intracytoplasmic sperm injection, or somatic cell nuclear transfer in domestic animal industries. However, retrieval of bovine oocytes after cryopreservation is not fully sufficient for subsequent embryo production. Likely obstacles for successful cryopreservation of bovine oocytes included the high volume/ surface ratio, depolymerization of spindle tubulin, abnormal aster formation, and premature release of cortical granules of metaphase-II stage (MII) oocytes (Hwang & Hochi, 2014). In addition, oocytes from domestic species, such as cattle and pigs, are known to be highly sensitive to cryoinjuries due to the high level of cytoplasmic lipid droplets when compared with human and rodent oocytes (Zhou & Li, 2013). Lcarnitine has been used to reduce intracellular lipids and improve the cryotolerance of bovine MII oocytes (Chankitisakul et al., 2013;Sprícigo et al., 2017). During the past decade, anti-oxidative chemicals, such as α-tocopherol (Yashiro et al., 2015), melatonin (Zhao et al., 2016), and resveratrol Sprícigo et al., 2017), were also applied to improve oocyte cryotolerance.
Vitrification (Rall & Fahy, 1985), as a replacement for conventional freezing (Whittingham et al., 1972;Wilmut & Rowson, 1973), offered a simple, cost-effective, and efficient protocol in embryo cryopreservation, by the formation of an amorphous glass state rather than detrimental ice crystals. This protocol includes the dehydration of embryos by exposure to a concentrated vitrification solution (VS), followed by direct immersion of 0.25-ml plastic straw containers into liquid nitrogen (LN 2 ; 2,000-2,500 C/min), rather than dehydration during a slow cooling process in a conventional freezing regimen (0.3-2 C/min). Technicians are familiar with using plastic straw for embryo vitrification, because plastic straws have been used for bovine semen freezing/artificial insemination (0.5-ml volume) and embryo slow freezing/transplantation (0.25-ml volume). Traditional straw vitrification does not require specialized freezing equipment for a constant rate of slow cooling, but the time allowed for exposure to highly toxic VS is quite short (approximately 1 min), resulting in a tight time schedule and a decreased sample number per single operation, as well as increased pressure upon technicians. The use of straw vitrification for bovine oocytes remained inefficient, despite of a 9% blastocyst yield (calculated from the total number of cryopreserved oocytes unless specified hereafter) and the in vivo development to two fetuses from post-warmed oocytes reported in an early study (Hamano et al., 1992). This article provides an up-to-date review of cryodevices suitable for minimum volume cooling (MVC) vitrification of bovine oocytes at the immature germinal vesicle (GV)-and mature MIIstages.

| CRYODEVICES FOR OOCYTE MVC VITRIFICATION
A significant breakthrough for the vitrification of bovine oocytes was achieved by the MVC procedure. Typical cryodevices designed for the MVC vitrification of bovine oocytes are illustrated in Figure 1. A 15% blastocyst yield was resulted from post-warmed MII oocytes vitrified with a MVC protocol using an electron microscope (EM) copper grid as the cryodevice (Martino et al., 1996). The EM grid was originally adopted to cryopreserve chilling-sensitive Drosophila melanogaster embryos, which had a mm-scale diameter (Mazur et al., 1992;Steponkus et al., 1990). The MVC vitrification protocol involved a 3-15 min exposure of oocytes to equilibration solution containing moderate concentrations of membrane-permeating cryoprotective agents (CPAs; dimethylsulfoxide [DMSO] and/or ethylene glycol [EG]; 3-20%) and a 1 min exposure to VS containing higher concentrations of the permeable CPAs (30-40%) and a non-permeating disaccharide (sucrose or trehalose) before rapid cooling to LN 2 temperature. Within the short exposure time to the VS, oocytes must be placed into or onto a cryodevice with a minimal volume of VS to accelerate the cooling rate. There is no strict definition of the "minimum" volume for bovine oocyte vitrification, but it is generally considered to be less than 1 μl. After storage in LN 2 and rapid warming, CPAs are removed from the oocytes in a stepwise manner using decreasing concentrations of sucrose solution. Various types of cryodevices have been developed to accelerate the cooling rate of bovine oocytes and can be theoretically divided into two categories: tubing and surface devices (Saragusty & Arav, 2011). Completely device-less protocols have also been applied to the MVC vitrification of bovine mature oocytes, with a blastocyst yield of 9% in a solid surface vitrification (SSV) system (Dinnyés et al., 2000) and 30% in microdrop (MD) method (Papis et al., 2000). A brief list of blastocyst yields from vitrified-warmed bovine MII oocytes using different crydevices is shown in Table 1.
Tubing device: Oocytes are aspirated into a tubing-type cryodevice. Vajta et al. (1998) reported that a very high cooling rate (>20,000 C/min) can be achieved in an open pulled straw (OPS), F I G U R E 1 Cryodevices used for oocyte MVC vitrification. Tubing-type: open pulled straw (OPS) and hollow fiber (HF). Surface-type: electron microscope (EM) grid, Cryoloop, Cryotop, triangle nylon mesh (NM), and multilayer silk fibroin (SF) sheet resulting in a 13% blastocyst yield from post-warmed bovine MII oocytes and a calf birth, followed by a few minor modifications as superfine OPS (Isachenko et al., 2001) and sealed pulled straw (Chen et al., 2001). Conventional disposable plastic tools were successfully used for the MVC vitrification of bovine oocytes, as reported with the gel-loading tip (Tominaga et al., 2005) or flexipet-denuding pipette (Morat o et al., 2008). Because of the big market for human ART, a closed system using CryoTip ® (Fujifilm Irvine Scientific) has been commercialized and is widespread in infertility clinics (Kuwayama, Vajta, Ieda, et al., 2005;VerMilyea & Brewer, 2017). Sanitary cryopreservation by the closed system is desired to avoid the possible crosscontamination of vitrified oocytes with pathogens within the LN 2 tank, but its importance may be dependent of the target animal species. Recently, hollow fiber vitrification (HFV) has been applied to bovine mature oocytes, with a 23% blastocyst yield from postwarmed oocytes (Kornienko et al., 2020). The triacetate cellulose hollow fibers (HF; 200-μm inner diameter, 15-μm-thick) used in the above study are enriched with 7-nm pores in the walls. The HFV system is attractive in terms of increased sample number per device and medium exchange without aspiration-based collection of the oocytes Matsunari et al., 2012).

| SMART DEVICES FOR BULK OOCYTES AND SELF-VS ABSORPTION
Oocyte numbers loaded per cryodevice are limited because of the strict requirement in minimizing the VS volume in a tight timetable (recommended quantity 10, possible upper limit 20, in MVC vitrification). Even in the HFV system, the maximum number per single HF was reported to be 12-17 in bovine oocytes (Kornienko et al., 2020) and 20 and 40 in porcine and murine oocytes, respectively . Such a limitation does not cause inconvenience in the clinical cryopreservation of human oocytes, because one to three oocytes are routinely loaded per cryodevice. For bovine species, all oocytes retrieved from a single donor (a pair of ovaries) may be T A B L E 1 MVC vitrification of bovine mature (MII) oocytes using different cryodevices and blastocyst generation following IVF  Matsumoto et al. (2001) first reported that as many as 65 bovine immature oocytes can be vitrified-warmed in bulk using nylon mesh (NM) as a cryodevice. Although no blastocysts were obtained in this early study, the same group achieved an 8% blastocyst yield and a live calf from post-warmed immature oocytes after a few modifications of the equilibration treatment (Abe et al., 2005).
Our laboratory designed a triangle NM sheet ( (Momozawa et al., 2017(Momozawa et al., , 2019. Tracing paper was also used as the VS-absorbable cryodevice in the MVC vitrification of bovine oocytes (Paul et al., 2018). Further improvement of the high absorption material and holding tool would help its practical application, such as the commercial KVS product Diamour (Mitsubishi Paper Mills Ltd, Tokyo, Japan).

| APPLICATION TO IMMATURE OOCYTES
Research for the cryopreservation of immature oocytes is important because of the increasing demands to retrieve human immature oocytes in cases of ovarian hyper-stimulation syndrome or cancer in young patients (Yamanaka et al., 2007).  (Hyttel et al., 2000). Transmission EM observation indicated that hyperosmotic VS conditions and ultrarapid cooling during vitrification procedures had a detrimental impact on the functional integrity of gap junctions in domestic species (Fuku et al., 1995;Hochi et al., 1996). Some reports described the effect of full or partial denuding of bovine COCs on their cryotolerance ( Figure 2). After Cryotop vitrification at the GV stage, Zhou et al. (2010) found 11% and 4% blastocyst yields from bovine full-size COCs and partially denuded COCs, respectively. In contrast, we showed a positive effect of downsizing cumulus cell layers on cryotolerance after Cryotop vitrification, with 14%, 18%, and 8% blastocyst yields from bovine full-size COCs, downsized COCs, and denuded oocytes, respectively (Tashima et al., 2017).
Ultra-rapid cooling rates have been achieved by direct immersion of oocytes/cryodevice into cryogenic LN 2 (À196 C) with a little boiling at first contact. A physical phenomenon "Leidenfrost effect," which refers to the quick development of nitrogen gas bubbles, generates a pocket of nitrogen vapor around oocytes and results in delay of heat transfer through its insulator-like action. Vitrification in N 2 slush (a mixture of solid and liquid nitrogen) has been considered as a new strategy to increase the cooling rate of oocytes (Santos et al., 2012), because it can avoid the Leidenfrost effect. However, Martino et al. (1996), the pioneers of oocyte EM grid vitrification, reported a comparable or rather inferior blastocyst yield from bovine MII oocytes vitrified in N 2 slush (À207 C) versus LN 2 (À196 C). In OPS vitrification of bovine immature oocytes, one Chinese group published the suitability of liquid helium (À269 C) as an alternative to conventional LN 2 (blastocyst yield 10% vs. 5%, Yu et al., 2016;13% vs. 9%, Xu et al., 2017;13% vs. 1%, Zhang et al., 2020). Interestingly, the immature oocytes vitrified in liquid helium had fewer intracytoplasmic lipid droplets after IVM compared with those vitrified in LN 2 (Xu et al., 2017). Our laboratory also observed unique kinetics  improving the cryotolerance of bovine immature oocytes is to eliminate DMSO from the VS, because this traditional CPA has a higher chemical toxicity than EG or propylene glycol (PG) (Awan et al., 2020).
An equal proportion of EG and DMSO has been long considered the most efficient combination of permeating CPA in VS. The membrane permeability of DMSO is lower than that of EG, probably because of the involvement of different aquaporin channels (Edashige, 2016

| CONCLUSIONS
This article focused on providing an up-to-date review of cryodevices used for MVC vitrification of bovine mature/immature oocytes in the past two decades, noting that a recent article has comprehensively summarized the bovine oocyte vitrification (Dujíčková et al., 2021). ments. Understanding and further improvement of cryodevicedependent performance for oocyte survivability will significantly contribute to efficient embryo production by IVM/IVF, in cattle breeding industries and also in human ART.

ACKNOWLEDGMENTS
The author thanks Dr. Masumi Hirabayashi (National Institute for Physiological Sciences, Aichi, Japan) for providing critical comments, Mr. Ryo Naito (Shinshu University, Nagano, Japan) for drawing cryodevice illustrations, and Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. This work is realized with research funding from the Japan Society for the Promotion of Sciences (JSPS; 16K07985, 20K06364, and 21H01783).

CONFLICT OF INTEREST
The author declares no conflict of interest.