Cryopreservation Competence of Chicken Oocytes as a Model of Endangered Wild Birds: Effects of Storage Time and Temperature on the Ovarian Follicle Survival

Simple Summary Many avian species are classified as endangered, and the development of the gamete preservation technique is essential to preserve their genetic diversity. Since cryopreservation and transplantation of ovarian tissues are currently the only methods of female fertility preservation in birds after birth, transporting the ovary to the laboratory in good condition is key for successful female fertility preservation. Here, we report using chickens as a model of wild birds for which storage at low temperature (4 °C), but not room temperature, can maintain the ovary, ensuring oocyte survival, morphology and protein expression of germ cell markers for at least 3 days. Furthermore, a vitrification study confirmed that oocytes after ovary storage at a low temperature can be preserved by ovarian tissue cryopreservation. On the other hand, ovaries stored at room temperature decreased oocyte survival, while increasing gene expression of apoptosis and oxidation stress markers. Our study not only verifies the fact that the storage of hen ovaries at low temperatures protects oocytes, which can be preserved by vitrification, but it also contributes to understanding the effect of ovary storage on the integrity and cryotolerance of immature follicles within the hen ovary. Abstract For the conservation of endangered avian species, developing gamete preservation technologies is essential. However, studies in oocytes have not been widely conducted. In this study, assuming that the ovaries are transported to a research facility after death, we investigated the effect of ovary storage on oocytes for the purpose of cryopreserving avian female gametes by using a chicken as a model of endangered avian species. After excision, the ovaries were stored at either a low temperature (4 °C) or room temperature for 1–3 days. Ovarian follicles stored under different conditions for each period were examined by neutral red staining, histology, and gene and protein expression analysis. In addition, the pH of the storage medium after preserving the ovaries was measured. Then, ovarian tissues were vitrified to determine the cryopreservation competence. Storing the ovarian tissues at 4 °C kept the follicles viable and morphologically normal for 3 days with slow decline. In contrast, although different storage temperature did not influence follicle viability and morphology after only 1 day of storage, ovarian tissues stored at room temperature rapidly declined in structurally normal follicles, and viable follicles were rarely seen after 3 days of storage. Gene and protein expression analysis showed that apoptosis had already started on the first day, as shown by the higher expression of CASP9 under room temperature conditions. Furthermore, high expression of SOD1 and a rapid decline of pH in the storage medium under room temperature storage suggested the influence of oxidative stress associated with low pH in this condition on the follicle survivability in hen ovarian tissues. Our cryopreservation study also showed that ovarian tissues stored at 4 °C could recover after cryopreservation even after 3 days of storage. The described storage conditions and cryopreservation methods, which preserve chicken follicle survival, will lay the foundation of ovarian tissue preservation to preserve the fertility of wild female birds.

Introduction
The International Union for Conservation of Nature's (IUCN's) Red List of Threatened Species warns that as many as 13% of birds are listed as endangered [1]. In this respect, gamete preservation plays an important role in the conservation of genetic diversity and creating reproductive opportunities for more individuals of endangered species. However, research in avian gamete preservation has gained less attention than that in mammals. This is essential for the preservation of species, especially of endangered birds, such as the Okinawa rail (Gallirallus okinawae), which is an endangered endemic species in the northern part of Okinawa Island, Japan [2]. For the development of genetic preservation technology for avian species, the primordial germ cells (PGCs), which are progenitor germ cells in the embryonic stage, are widely studied for the purpose of conservation of avian genetic resources [3]. While the preservation of PGCs is a promising technique in combination with chimera generation, the feasibility of this strategy for genetic conservation is limited, especially for conservation purposes, because it can be useful only in specific cases, when eggs that do not hatch are obtained. Alternatively, research on semen freezing has been actively carried out in domestic avian species for poultry production. More recently, semen cryopreservation has started to be studied in non-domestic birds, such as endangered cranes [4]. Meanwhile, the cryopreservation of female germplasm has been challenging in females due to the characteristics of avian ovaries and eggs with a large amount of yolk, which prevent the application of the cryopreservation techniques that have been used for mammals.
The chicken has a single ovary with different-sized follicles, which enclose primary oocytes, including a primordial follicle, primary follicles, and a pre-hierarchical follicle stage. While cryopreservation of ovarian tissue has been studied in mammals as a potential method to preserve female fertility, including in women threatened by cancer therapies [5], it has been also attempted in hens [6,7] and Japanese quail [8,9]. Moreover, some studies have revealed that ovarian tissue cryopreservation can be of use for transplantation, resulting in successful birth, for genetic preservation purposes [8,10,11]. Therefore, ovarian tissue cryopreservation is a reliable technique for the preservation of female genetic resources of endangered avian species. Since the combination techniques of cryopreservation and transplantation of ovarian tissues are currently the only methods of female fertility preservation in birds after birth, it is essential to cryopreserve the ovarian tissue in good condition.
Traffic accidents are a common threat for wild birds, including endangered species such as Okinawa rail, and there is a need to take the opportunity of obtaining the reproductive tract and germplasm in situ to use for conservation purposes [12]. In general, habitats of wild endangered birds are far from the laboratory. To recover their germplasms to use for cryopreservation, ovaries need to be stored in a good condition for a long period of time until ovarian tissues are cryopreserved. In mammals, the impact of different storage times and temperatures on the quality of oocytes is different among species. For instance, the maturation rates of oocyte nuclei decrease with the increase in ovary storage time in pigs [13], but do not change in horses [14]. The storage temperature of the ovary for transportation affected oocyte meiotic competence in vitro in cats [15], dogs [16] and pigs [13] and changed the cleavage and blastocyst formation rates in bovines [17]. However, little information is available regarding the effects of ovary storage conditions on ovarian tissue cryopreservation. And moreover, to our knowledge, no reports have been published on the influence of ovary storage conditions in avian species.
The overall goal of the present study was to understand appropriate shipping/storage conditions for avian ovaries that can sustain female gamete preservation in birds. By using ovaries of laying hens (Gallus gallus domesticus) as a model animal of endangered avian species, we investigate different storage conditions for the purpose of cryopreserving avian using ovaries of laying hens (Gallus gallus domesticus) as a model animal of endangered avian species, we investigate different storage conditions for the purpose of cryopreserving avian ovarian tissues. Our specific objectives were to determine the influences of (i) storage temperature (low (4 °C) vs. room temperature) and (ii) the storage period (1, 2 and 3 days) on the viability and morphology of follicles within hens' ovaries as well as gene/protein expression. Cryopreservation experiments were also carried out to evaluate if the storage methods optimally maintain immature follicles within the chicken ovarian tissues.

Chemicals
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated.

Collection of Chicken Ovarian Tissues
The ovaries of laying hens of White Leghorn breed (Gallus gallus domesticus) (1 year old; n = 30) maintained at the Nippon Veterinary and Life Science University (Tokyo, Japan) were used. Ovaries were removed immediately after sacrifice by decapitation, rinsed with PBS, and immersed in storage medium of L-15 containing 10 mM HEPES, 100 μg/mL streptomycin sulphate (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 100 μg/mL penicillin G sodium (FUJIFILM Wako Pure Chemical Corporation), and 0.5 mM Ascorbic acid (pH 7.1) [18,19]. Ovaries in the storage medium were then transported to the laboratory either at 4 °C or room temperature and stored under the same conditions as the shipping temperature for 1-3 days until use. The pH of the storage medium was measured at the end of the storage in each ovary.
Ovaries were rinsed and then dissected in working medium of Hanks' MEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 15 mM Hepes, 1% BSA, 2 mM glutamax (Thermo Fisher Scientific), 100 μg/mL streptomycin sulphate (FUJIFILM Wako Pure Chemical Corporation), and 100 μg/mL penicillin G sodium (FUJIFILM Wako Pure Chemical Corporation) [18]. Follicles larger than 2 mm were mechanically removed from the ovaries ( Figure 1A) and tissues were cut into equal pieces (5 mm 2 ). Because the follicles are not evenly distributed in the ovaries, at least two pieces of ovarian tissues from each donor stored under various conditions were utilised for each experiment. In some of the ovaries stored at 4 °C for different periods, the remaining ovarian tissues were vitrified.

Neutral Red (NR) Staining to Assess Viable Ovarian Follicles in Fresh and Vitrified-Warmed Tissues
At least two pieces of fresh or vitrified-warmed ovarian tissues were rinsed in MEM working medium and then incubated in 0.5 mL of enzyme solution consisting of 1.5 mg/mL collagenase and trypsin/ETDA in MEM working medium in the 5% CO 2 incubator for 2.5 h at 38.5 • C with gentle agitation. The enzymatic digestion of ovarian tissues was stopped by addition of the KAv-1 medium, MEM-based medium containing 5% fatal bovine serum and 5% chick serum [20]; then, ovarian tissues were incubated with Kav-1 medium supplemented with 33 µg/mL of NR solution (2-amino-3 methyl-7-dimethylaminophenazoniumchloride) in the CO 2 incubator for 2 h at 38.5 • C. Follicles consisting of an oocyte surrounded by granulosa cells were easily distinguished from stromal cells within the ovarian tissues after incubation with enzyme solution. The number of redcoloured follicles stained with NR (viable) and uncoloured (dead) follicles were counted in each ovarian tissues under a stereomicroscope (Nikon SMZ800N, Nikon, Tokyo, Japan). Follicles were only considered to be viable when both the oocyte and more than 75% of the surrounding granulosa cells stained positive for NR ( Figure 1B).

Histological Assessment and Classification of Follicular Structure
Histological assessment and classification of follicle structure were conducted as described previously with slight modification [18,21]. Briefly, at least three pieces of ovarian tissue in each group were fixed with Bouin's solution (FUJIFILM Wako Pure Chemical Corporation), kept at 4 • C overnight, dehydrated in a series of ethanol solutions (70-100%), and embedded in paraffin. Serial sections (5 µm thick) of ovarian tissues were prepared and stained with haematoxylin and eosin (both Muto Pure Chemicals CO. Tokyo, Japan). To avoid double counting, three sections, each at least 20 µm apart, were assessed by light microscopy (Nikon ECLIPE E600, Nikon).
Follicles within the ovarian tissues were classified as primordial (<80 um), primary (0.08-1 mm), or pre-hierarchical (>1 mm) [8]. All the follicles were further characterized as follows: 'normal', when the layer of granulosa cells is attached to the spherical oocyte surrounding it and the homogenous ooplasm contains a tiny granulated nucleus, or 'abnormal', wherein aggregation and shrinkage of nuclear chromatin and wrinkling of the nuclear membrane were regarded as signs of atresia [6]. The proportion of morphologically normal follicles per section was calculated by dividing the number of normal follicles by the total number of assessed follicles.

Gene Expression Analysis by Quantitative Reverse Transcription Polymerase Chain Reaction (qPCR)
The expression of caspase 3 (CASP3) and 9 (CASP9) as well as superoxide dismutase 1 (SOD1) was determined by qPCR using ovaries under different storage conditions. Two to three tissues recovered from ovaries stored at different temperatures for 1-3 days were soaked in RNAlater (Invitrogen, Carlsbad, CA, USA) and stored at −80 • C until RNA extraction. After thawing, RNA was extracted by using the RNeasy Mini kit (QIAGEN, Hilden, Germany) and reverse transcription was performed with a Prime Script RT-PCR kit (Invitrogen), both according to the manufacturer's instructions. For each sample, qPCR was completed in triplicate using SYBR Pemix Ex Taq II (TaKaRa, Shiga, Japan) and a Thermal Cycler Dice Real-Time System TP800 (TaKaRa). PCR amplification was conducted with 40 cycles (95 • C for 5 s and 60 • C for 30 s). Amplification and melting curves of each PCR product were checked to verify efficiencies and the targeted amplicon. The CT value of each gene was normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, housekeeping gene). The qPCR data expressed as the relative mRNA expression and fold change over ovaries stored at room temperature for 1 day were analysed with the machine's software (Thermal Cycler Dice Real Time System TP800 Software, ver. 5.11B, TaKaRa). The primers for specific transcripts were designed using the Primer3 software, with all the sequences listed in Table 1.

Vitrification and Warming Procedure
The ovarian tissue vitrification was carried out using a needle immersion vitrification protocol described previously [9] with slight modification. Briefly, ovarian tissues were threaded onto a 27 G needle (5-6 pieces per needle; Terumo Corporation, Tokyo, Japan) with space between each piece. The needles that pierced the tissue were rinsed in KAv-1 medium at first and immersed in equilibration solution consisting of 7.5% dimethyl sulfoxide (DMSO) and 7.5% ethylene glycol (EG) in KAv-1 medium for 10 min at 4 • C. The needles were subsequently moved into vitrification solution consisting of 15% DMSO, 15% EG, and 0.5 M sucrose in KAv-1 for 10 min at 4 • C. Then, after excess solution was absorbed quickly with a Kimwipe, the needles were plunged into liquid nitrogen directly, inserted into a cryovial (Watson, Tokyo, Japan) containing liquid nitrogen, and stored for at least 1 week.
For warming, needles holding tissue pieces were taken out of the cryovial in liquid nitrogen and immediately transferred into a washing gradient solution. The vitrified tissues held by the needles were washed in gradient solution (KAv-1 supplemented with 1, 0.5, 0.25, and 0 M sucrose) for 5 min at each step at 37 • C. The ovarian tissues were then removed from the needles for further assessment. The ovary from each animal was randomly allocated to one of two storage temperatures, either low (4 • C) or room temperature. Then, ovaries and storage medium at each storage temperature were examined at different storage periods (1, 2 and 3 days). Hence, we divided 30 hens into five replicates of six different storage conditions (three storage periods of room vs. low temperature). The effect of different storage temperatures and times on ovarian follicles were evaluated by pH analysis in storage medium (n = 5 per condition), then follicle normality by NR staining and histology (n = 4 per condition in each analysis, at least 2 pieces/hen/condition) in ovarian tissues that were separately stored under different conditions. Gene and protein expressions were then evaluated by qPCR and Western blotting (n = 3 per condition in each analysis, 2-3 pieces/hen/condition).

Study 2: Examine the Freezing Tolerance of Ovarian Follicles Stored for Different Storage Periods
Based on the results of Study 1, ovaries stored at low temperatures (4 • C) were vitrified for different storage periods (1, 2 and 3 days) to investigate the effect of storage time by vitrification on freezing tolerance of ovarian follicle. After warming, vitrified-warmed tissues were examined with NR staining and ovarian follicle histology (n = 4 per storage period in each analysis, at least 2 pieces/hen/storage period).

Statistical Analysis
Each value shown is the mean with the standard error of mean (SEM). The Shapiro-Wilk test was used to determine if the dataset was normal. Additionally, the Bartlett test was used to evaluate the homogeneity of variance. Analysis of variance (ANOVA) followed by a Turkey's multiple comparison test were applied to compare follicle viability and morphology, pH of storage medium and relative expression level of mRNA among different storage conditions or before/after vitrification. Differences were considered significant at a level of p < 0.05 (GraphPad PRISM 9, GraphPad Software, La Jolla, CA, USA).

Changes in pH of Storage Medium and Follicle Viability in Chicken Ovarian Tissues under Different Storage Conditions
A starting point of our analysis was to investigate the effect of different storage temperatures (low vs. room temperature) and different storage periods (1 to 3 days) on the pH of the medium in which the hen ovary was stored. While the pH of the storage medium did not change much after 1 day of storage in the low-temperature conditions (from pH 7.1 to pH 7.0), the pH value decreased greatly at room temperature (pH 6.7) compared to before storage and to that at the low temperature at 1 day of storage ( Figure 1C). At the low temperature, the pH of the storage medium was slightly decreased in a time-dependent manner, but the changes were maintained as minimal for 3 days of storage from pH 7.0 to pH 6.9. On the other hand, at room temperature, although the pH of the storage medium was maintained from day 1 to day 2 of storage at around pH 6.7, it dropped rapidly to pH 6.5 after 3 days of storage.
The viability of ovarian follicles stored under different conditions for each period was evaluated by NR staining ( Figure 1B). The viability of the follicles within the stored ovaries was also decreased in a time-and temperature-dependent manner. At 1 day after storage, both temperature groups had follicles with positive NR staining, which were considered to be viable. The difference in viability among different storage temperatures was small, and ovaries stored at a low temperature had slightly more viable follicles (76.1 ± 0.8%) compared to those at room temperature (66.5 ± 1.1%, Figure 2A). However, the difference in viability in ovarian follicles was increased as storage time increased. In the low-temperature group, although the viability of ovarian follicles was gradually decreased, 52.6 ± 1.3% of the follicles within the ovarian tissues still survived after 3 days of storage (Figure 2A). On the other hand, in the room-temperature group, the viability of ovarian follicles was already decreased to 42.0 ± 1.8% at 2 days of storage, and surviving follicles were rarely seen (3.5 ± 1.6%) after 3 days of storage (Figure 2A).

Influence of Different Storage Conditions on Gene and Protein Expressions in Chicken Ovarian Tissues
Gene expression analysis showed that expression level of CASP9, an apoptosis marker gene, tended to be higher in the room-temperature group compared to the lowtemperature group at the first day of storage (p = 0.56, Figure 3). In the low-temperature group, expression of CASP9 was maintained as low for 2 days of storage but became upregulated at 3 days. A different apoptosis marker gene, CASP 3, did not differ among Histological examination revealed that ovarian tissues examined for the current study were mostly (>98%) primordial and primary follicles and more than 80% of them were intact on the first day of storage in both temperature groups ( Figure 2B,C). However, at 2 days of storage, while the low-temperature condition maintained the proportion of intact follicles (81.2 ± 1.5%), ovarian tissues in the room-temperature condition tended to decrease the proportion of intact follicles to 48.5 ± 2.0% ( Figure 2B,C). The difference in follicle maintenance became significantly different (p = 0.01) after 3 days of storage. Although ovarian tissues stored under the low-temperature conditions still kept 60.0 ± 2.6% of intact follicles after 3 days of storage, intact follicles were rarely seen (3.7 ± 1.1%) in the roomtemperature conditions at the same storage period ( Figure 2B,C).

Influence of Different Storage Conditions on Gene and Protein Expressions in Chicken Ovarian Tissues
Gene expression analysis showed that expression level of CASP9, an apoptosis marker gene, tended to be higher in the room-temperature group compared to the low-temperature group at the first day of storage (p = 0.56, Figure 3). In the low-temperature group, expression of CASP9 was maintained as low for 2 days of storage but became up-regulated at 3 days. A different apoptosis marker gene, CASP3, did not differ among storage condition and time (Figure 3). The analysis of SOD1 expression, a marker of oxidation stress, indicated a trend of higher expression levels in the room-temperature group than in the low-temperature group (Figure 3). In both conditions, SOD1 expression tended to increase as storage time increased (Figure 3).
Animals 2022, 12, x FOR PEER REVIEW 9 of 15 storage condition and time ( Figure 3). The analysis of SOD1 expression, a marker of oxidation stress, indicated a trend of higher expression levels in the room-temperature group than in the low-temperature group (Figure 3). In both conditions, SOD1 expression tended to increase as storage time increased (Figure 3). Western blotting analysis revealed higher expression of CASP9 in the room-temperature group than in the cold group, especially on the first day of storage (Figure 4). On the other hand, CASP9 protein expression in the low-temperature group remained low for 3 days of storage (Figure 4). The expression of the germ cell marker, Chicken Vasa Homologue (CVH), was also determined in the ovarian tissues in both group for each storage time (Figure 4).

Influence of Storage Time on Cryopreservation of Chicken Ovarian Tissues
To understand how ovary preservation might affect the freezing tolerance of ovarian follicles, follicular viability and morphological maintenance were evaluated after ovarian tissue vitrification by NR staining and histological analysis. Based on the results of Study Western blotting analysis revealed higher expression of CASP9 in the room-temperature group than in the cold group, especially on the first day of storage (Figure 4). On the other hand, CASP9 protein expression in the low-temperature group remained low for 3 days of storage ( Figure 4). The expression of the germ cell marker, Chicken Vasa Homologue (CVH), was also determined in the ovarian tissues in both group for each storage time ( Figure 4).
Animals 2022, 12, x FOR PEER REVIEW 9 of 15 storage condition and time (Figure 3). The analysis of SOD1 expression, a marker of oxidation stress, indicated a trend of higher expression levels in the room-temperature group than in the low-temperature group (Figure 3). In both conditions, SOD1 expression tended to increase as storage time increased (Figure 3). Western blotting analysis revealed higher expression of CASP9 in the room-temperature group than in the cold group, especially on the first day of storage (Figure 4). On the other hand, CASP9 protein expression in the low-temperature group remained low for 3 days of storage (Figure 4). The expression of the germ cell marker, Chicken Vasa Homologue (CVH), was also determined in the ovarian tissues in both group for each storage time ( Figure 4).

Influence of Storage Time on Cryopreservation of Chicken Ovarian Tissues
To understand how ovary preservation might affect the freezing tolerance of ovarian follicles, follicular viability and morphological maintenance were evaluated after ovarian

Influence of Storage Time on Cryopreservation of Chicken Ovarian Tissues
To understand how ovary preservation might affect the freezing tolerance of ovarian follicles, follicular viability and morphological maintenance were evaluated after ovarian tissue vitrification by NR staining and histological analysis. Based on the results of Study 1, ovaries stored in low-temperature conditions for different storage times were utilized for vitrification (n = 4/storage time).
NR staining revealed that all of the vitrified ovarian tissues had follicles with positive NR staining, which were considered to be viable ( Figure 5A). The proportion of viable follicles within ovarian tissues significantly decreased after vitrification at each preservation time (p < 0.005, Figure 5A). Similar to the ovarian tissues before vitrification, the percentage of viable follicles tended to decrease in vitrified tissues as preservation time was increased, but approximately 20% of follicles still survived after vitrification in the ovarian tissues preserved for 3 days prior to vitrification. (p = 0.12, Day 1, 38.1 ± 2.1%; Day 2, 32.5 ± 2.2; Day 3, 19.6 ± 2.0%; Figure 5A).
1, ovaries stored in low-temperature conditions for different storage times were utilized for vitrification (n = 4/storage time).
NR staining revealed that all of the vitrified ovarian tissues had follicles with positive NR staining, which were considered to be viable ( Figure 5A). The proportion of viable follicles within ovarian tissues significantly decreased after vitrification at each preservation time (p < 0.005, Figure 5A). Similar to the ovarian tissues before vitrification, the percentage of viable follicles tended to decrease in vitrified tissues as preservation time was increased, but approximately 20% of follicles still survived after vitrification in the ovarian tissues preserved for 3 days prior to vitrification. (p = 0.12, Day 1, 38.1 ± 2.1%; Day 2, 32.5 ± 2.2; Day 3, 19.6 ± 2.0%; Figure 5A).
Histological observations of vitrified tissues showed the presence of structurally intact follicles with visually healthy nuclei in all the ovarian tissues regardless of the preservation time ( Figure 5B,C). Specifically, ovarian tissues preserved for 1 day retained 68.9 ± 1.5% of intact follicles after vitrification ( Figure 5B). This value did not significantly change as preservation time increased, but it tended to decrease as preservation time increased. Nevertheless, histological observation clearly showed that the ovarian tissues preserved over 2 days still hold the morphologically normal follicles (Day 2, 57.1 ± 2.7%; Day 3, 46.9 ± 2.2%. Figure 5B). Mean (± SEM) percentages of follicle viability after vitrification of ovarian tissues stored for different periods (for 1 to 3 days). * indicate statistically significant differences (* p < 0.005, ** p < 0.001) in the percentages of survived follicles. (B) Mean (± SEM) percentages of morphologically normal follicle in ovarian tissues vitrified after ovary storage for different periods (for 1 to 3 days). (C) Figure 5. Influence of storage periods on follicle viability after vitrification of ovarian tissues (A) Mean (± SEM) percentages of follicle viability after vitrification of ovarian tissues stored for different periods (for 1 to 3 days). * indicate statistically significant differences (* p < 0.005, ** p < 0.001) in the percentages of survived follicles. (B) Mean (± SEM) percentages of morphologically normal follicle in ovarian tissues vitrified after ovary storage for different periods (for 1 to 3 days).
(C) Histomicrographs of ovarian tissues vitrified after ovary storage for different periods stored at low temperature for 1 to 3 days). "a" indicates the abnormal follicles. Bar = 100 µm.
Histological observations of vitrified tissues showed the presence of structurally intact follicles with visually healthy nuclei in all the ovarian tissues regardless of the preservation time ( Figure 5B,C). Specifically, ovarian tissues preserved for 1 day retained 68.9 ± 1.5% of intact follicles after vitrification ( Figure 5B). This value did not significantly change as preservation time increased, but it tended to decrease as preservation time increased. Nevertheless, histological observation clearly showed that the ovarian tissues preserved over 2 days still hold the morphologically normal follicles (Day 2, 57.1 ± 2.7%; Day 3, 46.9 ± 2.2%. Figure 5B).

Discussion
This work demonstrates the relationship between oocyte viability and storage time and temperature in avian ovaries for the first time. Cryopreservation of ovarian tissue is a promising approach for female fertility preservation, and successful transportation of the ovary is a key factor for effective cryopreservation of avian female gonads as a means of genetic preservation. Using a chicken as a model for wild avians, this study was the first to evaluate the storage condition of ovaries for the purpose of ovarian tissue cryopreservation based on viability, morphology and gene/protein expressions of follicles and its cryotolerance in this species. Specifically, the present study demonstrates that a low (4 • C) temperature can successfully maintain the viability and morphology of primordial and primary follicles within hen's ovaries for 3 days. Furthermore, it was clear that follicles in the stored ovaries can survive after vitrification.
Trends in follicle viability affected by different storage conditions were similar between NR staining and histological analysis. In the present study, ovarian tissues stored at room temperature had lower cell viability and fewer morphologically normal follicles compared with their counterparts stored at a low temperature throughout the ovary storage. Furthermore, while the room-temperature condition did not retain follicle survival for 3 days of storage, low-temperature conditions maintained follicle survival and, moreover, cryotolerance determined by vitrification assay. A study in cats showed that ovary storage at 4 • C does not affect the meiotic competence of oocytes in vitro until 24 h of storage, but it decreases dramatically after 48 h of storage [15]. Here, our study demonstrated in hen ovaries that ovary storage at the same temperature surely decreased the follicle viability before and after vitrification in a time-dependent manner, but the decline did not occur rapidly compared to storage at room temperature. Moreover, especially in the room-temperature condition, we observed a decrease in the pH of the medium throughout ovary storage. In pig studies, decreasing pH was observed in the follicular fluid during ovary storage, with a more pronounced tendency at higher temperatures [24]. Coincidentally, follicle viability evaluated by NR staining and histological analysis decreased as pH decreased, therefore suggesting that oxidation stress caused the damage to ovarian follicles.
For the first time, the expression of some apoptosis-related genes and proteins in stored hen ovaries was measured. In general, caspase (cysteine-dependent aspartate-directed proteases) has a determinative role and activation in apoptosis. Activation of the caspase cascade results in cleavage and disintegration of substrate proteins and subsequent removal of the degenerating cell [25]. Our findings indicated that CASP9 was strongly expressed in the ovarian tissues stored at room temperature compared to the low-temperature group on the first day of ovary storage. While the highest expression of CASP9 was seen on the first day of storage in the room-temperature condition, it was seen on the third day in the low-temperature counterpart. Similarly, our Western blotting analysis showed high protein expression of CASP9 in the ovarian tissues stored at room temperature, especially on the first day of storage. Caspase 9 is known as an initiator of apoptotic signals, the same as caspase 2, 8, 10 [25]. Our result indicated that, while follicle viability decreased as storage time increased at both temperatures, intensive signalling of apoptosis and degeneration of cells in ovarian tissues was already started on the first day in the ovaries stored at room temperature. On the other hand, maintenance of a low expression level of CASP9 until the second day of storage in the low-temperature condition suggested that this condition minimized the effect of storage time on cell death of ovarian tissues. The superiority of low-temperature storage was also confirmed at the protein level: In ovaries stored at a low temperature, CVH expression was maintained, while CASP9 expression was kept low through the 3 days of storage. These results demonstrated that keeping the ovaries at a low temperature makes them less prone to apoptosis and maintains their function as germ cells. In contrast, it was shown that storage conditions did not change the expression of CASP3. This is unexpected because caspase 9 is known to directly cleave and activate caspase 3 [25]. On the other hand, caspase 3 also has other activators, including caspase 8 and 10 as well as caspase 12, each of which is induced separately and has distinct roles in apoptosis. For instance, while caspase 12 is induced by endoplasmic reticulum (ER) stress, caspase 8 is activated by dimerization and self-cleavage at the death-inducing signalling complex with caspase 10, and both activate apoptosis through caspase 3 [25]. On the other hand, caspase 9 is a mitochondrial apoptotic mediator and is induced by a mitochondrion-centred cell death that is mediated by mitochondrial outer membrane permeabilization (MOMP), resulting in increased production of reactive oxygen species (ROS) following cytochrome c release [25,26]. The generation of ROS can provoke damage to multiple cellular organelles and processes, which can ultimately disrupt normal physiology [27]. In a pig study, ovary storage for longer than 2 h at 25 • C and 35 • C resulted in the low pH of the follicular fluid and high ROS level in immature oocytes [24]. In the present study, decreases in pH in the storage medium occurred prior to the increase in CASP9 and matched the decrease in follicular integrity. Moreover, the expression of SOD1, the oxidation stress marker, tended to be higher in the room-temperature group than the low-temperature counterpart. Therefore, it is possible that production of ROS was induced by oxidation stress through the activation of caspase 9, resulting in the death of follicles in stored ovarian tissues. Because it is possible that oxidation stress is one of the main factors causing the degeneration of follicles during ovary storage, it would be worthwhile to investigate the changes in ROS within the stored ovaries and also the effect of antioxidants in the storage medium on follicle survival in the future.
It is worth noting that the influence of different storage temperatures was minimal in terms of follicle viability and morphology on the first day of ovary storage. This result was attractive, especially for conservation purposes of endangered avians. For example, in Okinawa rail, the body is not found immediately after death in their habitat and is left at room temperature until found, and then, in most cases, the ovaries are not recovered until at least 1 day after death. Although the environment surrounding the ovaries varies greatly depending on whether they are inside or outside of the body, and our gene/protein expression analysis and changes in the pH of the storage medium showed that the negative influence of room temperature has already started 1 day after storage as well as in the environment, it is possible that there is still a chance to recover the surviving follicle within the ovaries of birds left at room temperature for 1 day.
The vitrification study demonstrated that our storage protocol at low temperatures is suitable for maintaining the follicle integrity, including cryotolerance. To our knowledge, this is the first study to evaluate the effect of the storage period on follicle integrity after vitrification in avian ovaries. Since oocyte physiology is highly species-specific and commonly used cryoprotectants can also cause cell damage due to toxicity [28,29], choosing the proper agents and concentrations to reduce their toxicity is key in the development of a successful vitrification protocol of ovarian tissue. In this study, the use of KAv-1, specifically adjusted for avian cells with chick serum [20], resulted in a similar morphology normally compared to that of hen ovarian tissues in each storage period, suggesting that our freezing protocol could be effective for avian ovarian tissue vitrification. Our result is consistent with other vitrification studies in hen ovary [6], which successfully maintained follicle morphology with similar vitrification solutions except for the presence of chick serum in our study. The same group also reported the further improvement in follicle viability of hen ovarian tissues by supplementation of dietary polyunsaturated fatty acid and vitamin E as additional cryoprotectants [7]. A study in Japanese quail by another group showed that cell viability (but not follicle viability) in vitrified ovarian tissues were evaluated by trypan blue [8]. In our current study, while viability of ovarian cells was decreased after ovarian tissue vitrification, the authors successfully produced offspring from cryopreserved ovarian tissues by transplantation [8]. The difference between these previous studies and our study is that our study evaluated the ovarian tissue vitrification by not only histological observation or ovarian cell survival, but also by follicle viability using the NR staining assay. Although NR staining in the present study demonstrated that the ovarian tissues vitrified after ovary storage decreased the follicle viability, it also revealed the maintenance of follicle survival in vitrified ovarian tissues even after 3 days of ovary storage, suggesting the possibility of application for future transplantation to generate offspring. To increase follicle viability in vitrified hen ovaries, further improvement of the vitrification protocol is essential for applications of avian female fertility preservation. Nevertheless, our storage and vitrification protocol can serve as a useful shipping and cryopreservation method for avian ovaries to preserve follicle integrity, and future improvements to this system will be made. This is the first study to evaluate the viability of avian ovarian follicles by NR staining, and we also validated the value of performing NR staining to confirm follicular survival in ovarian tissues. Although trends of decrease in follicular integrity by increase in the storage period were the same between before and after vitrification in hen ovarian tissues, the difference between before and after vitrification was significant throughout the storage period when analysed by NR staining. This is unexpected because the mean percentage of follicle integrity evaluated by NR staining and histology was similar in the ovarian tissues before vitrification. It could be suggested that changes in follicular integrity cannot be detected based on histological analysis. This result is consistent with our previous study in vitrified canine ovaries. In this study, the xenotransplantation assay revealed the importance of evaluating the long-term follicle integrity by evidence of a loss of follicle integrity in the frozen tissues considered to have morphologically normal follicles by histological observation [30]. In avian species, at least to our knowledge, there are no studies about in vitro culture nor xenotransplantation of ovarian tissues, and it is difficult to assess the long-term survival of ovarian follicles except for orthotopic ovarian transplantation. Therefore, NR staining provides a useful assay for evaluating the follicle integrity in avian ovarian tissues.

Conclusions
In conclusion, immature follicles in the stored chicken ovaries can survive for 3 days at 4 • C, but not at room temperature. The vitrification study proved that some follicles within chicken ovarian tissues can be cryopreserved even after 3 days of storage at 4 • C. Our study verifies the fact that the storage of hen ovaries for long periods of time and at low temperatures protects follicles that can be preserved by vitrification, but it also contributes to elucidating the effect of ovary storage on the integrity and cryotolerance of immature follicles within the hen ovary. Our study could provide a promising shipping protocol for ovaries for female gamete preservation in wild endangered avian species.


CRISPR/Cas-Based Biosensor As a New Age Detection Method for Pathogenic Bacteria

Methods enabling rapid and on-site detection of pathogenic bacteria are a prerequisite for public health assurance, medical diagnostics, ensuring food safety and security, and research. Many current bacteria detection technologies are inconvenient and time-consuming, making them unsuitable for field detection. New technology based on the CRISPR/Cas system has the potential to fill the existing gaps in detection. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a part of the bacterial adaptive immune system to protect them from intruding bacteriophages. The immunological memory is saved by the CRISPR array of bacteria in the form of short DNA sequences (spacers) from invading viruses and incorporated with the CRISPR DNA repeats. Cas proteins are responsible for triggering and initiating the adaptive immune function of CRISPR/Cas systems. In advanced biological research, the CRISPR/Cas system has emerged as a significant tool from genome editing to pathogen detection. By considering its sensitivity and specificity, this system can become one of the leading detection methods for targeting DNA/RNA. This technique is well applied in virus detection like Dengue, ZIKA, SARS-CoV-2, etc., but for bacterial detection, this CRISPR/Cas system is limited to only a few organisms to date. In this review, we have discussed the different techniques based on the CRISPR/Cas system that have been developed for the detection of various pathogenic bacteria like L. monocytogenes, M. tuberculosis, Methicillin-resistant S. aureus, Salmonella, E. coli, P. aeruginosa, and A. baumannii.

INTRODUCTION
Rapid, sensitive, specific, and on-site detection of pathogenic bacteria is critical in clinical diagnosis, treatment, surveillance of foodborne disease, and biological research. 1−3 It helps to gather clinical information in order to provide appropriate treatment and prevent the spread of disease. 4 According to the World Health Organization's standard, an ideal pathogen diagnostic test should be assured: affordable, sensitive, specific, easy-to-use, rapid, without large equipment, and delivered to the user. 5 In order to detect nucleic acid signatures of pathogens, a vast array of detection methods have emerged based on PCR/qPCR, isothermal amplification-based detection assays, and next-generation sequencing. 6−8 To improve sensitivity, affordability, simplicity, and rapidity there are various advanced nucleic acid detection techniques developed so far. One of the latest and advanced methods is clustered regularly interspaced short palindromic repeats (CRISPR) associated systems (CRISPR/Cas), 9,10 which have recently gained great importance and attention in nucleic acid analysis and detection. In order to achieve higher sensitivity, the CRISPR/Cas system is frequently associated with polymerase chain reaction (PCR) and with isothermal nucleic acid amplification techniques like NASBA, 11 RCA, 12−14 SDA, 15,16 LAMP, 17 RPA, 18 and EXPAR. 19 The combination of CRISPR/ Cas with advanced isothermal amplification technologies is promoting the development of novel optical and electrochemical biosensing devices.
CRISPR/Cas systems provide adaptive protection to bacteria and archaea against invading foreign nucleic acids. 9,10 The CRISPR/Cas system in bacteria recognizes and degrades foreign genetic elements generally from viruses. 20 These systems are primarily guided by an RNA called guide-RNA (gRNA) or CRISPR RNA (crRNA), which recognizes the target and directs Cas proteins to locate and cleave invading DNA sequences. 21 This system works through three steps: adaptation or spacer-acquisition, 22 crRNA processing, 23 and interference. 24 In the spacer acquisition step, when new foreign DNA/RNA is introduced in the bacterial cell, a short piece of the DNA/RNA segment, called protospacer in the immediate upstream vicinity of a protospacer adjacent motif (PAM) present in the foreign DNA/RNA, is excised out by the help of the Cas1−Cas2 complex. This protospacer was then inserted as a new spacer into the bacterial genomic region of the CRISPR array ( Figure 1a) where all the acquired spacer resides. 22,25 The second step is crRNA biogenesis, which occurs when pre-CRISPR RNA (pre-crRNA) is transcribed by RNA polymerase (RNAP) from the CRISPR array region, then cleaved by specific endoribonucleases into small mature crRNA (Figure 1a). Each crRNA contains one complementary sequence of a spacer. 26,27 The final step is interference, 24 which entails sequence-specific targeting and cleavage of foreign DNA/RNA having a protospacer that is complementary to the spacer sequence in crRNA. To commence crRNA-mediated DNA binding, a protospacer adjacent motif (PAM) must be present in the immediate vicinity of a protospacer sequence. 28 crRNAs recognize and produce complementary base pairs unique to foreign RNA or DNA, resulting in the cleavage of the crRNA-foreign nucleic acid complex 29,30 (Figure 1a).
CRISPR/Cas systems are classified according to their utilization of specific Cas enzymes and methods of interference. In accordance with recent publications, CRISPR/Cas systems can be categorized into two classes: class 1 and 2, six types: types I−VI, and numerous subtypes. 31 Class 1 comprises types I, III, and IV; and Class 2 includes types II, V, and VI ( Figure 1b). Each type is distinguished by discrete effector module configurations that include various signature proteins. 32 The most widely used toolbox for nucleic acid detection belongs to the class 2 system that contains Cas9, Cas12, Cas13, and Cas14. 33 Cas9 (type II) and Cas12 (type V) target DNA, while Cas13 (type VI) targets RNA 34 and Cas14 targets ssDNA. 33 CRISPR/Cas systems, specifically Cas9 (type II), have become a popular tool for transcription regulation, genome editing, and in situ DNA/RNA detection in recent years. 35 Cas12 and Cas13 effectors have a unique property called "collateral cleavage". 36 In the presence of a target or reporter DNA/RNA, these Cas effectors are activated and can do collateral (nonspecific) cleavage on any singlestranded DNA/RNA present in the near vicinity. 37 The advantage of this collateral cleavage is that it can easily be detected by fluorescence reporters tagged in single-stranded DNA/RNA. This has recently displayed remarkable potential in developing novel biosensing technologies for nucleic acid detection. 37 This technology is widely harnessed for the detection of viral diseases, such as specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) 38−40 to detect Zika and Dengue 38 and DNA endonuclease-targeted CRISPR Figure 1. Basic understanding of CRISPR/Cas system and its classification. (a) CRISPR loci consisted of short repeats (gray boxes) interspaced by various short spacers (colored circular). CRISPR loci are flanked by a cluster of CRISPR-associated (Cas) genes (blue arrows) that encode proteins responsible for the different stages of the CRISPR/Cas system in adaptive immunity. When a bacteriophage (i) enters the cell, a small piece of invader DNA (protospacer) is added to the host chromosome's CRISPR array as a new spacer (red circle). Transcription takes place across the CRISPR array to generate pre-crRNA, which is then processed into short mature crRNAs. Each crRNA sequence contains one spacer and one flanking repetition. The effector complexes formed by the assembly of the crRNAs and Cas proteins serve as antisense guides for the effector complexes. When the same bacteriophage (ii) will attack the bacteria again, the existing crRNA having the spacer sequences complementary to the portion of the bacteriophage genome will degrade the phage genome by the CRISPR/Cas system. (b) There are two classes and six types of CRISPR/Cas systems where Cas3, Cas9, and Cas12 target the DNA, Cas13 targets RNA, and Cas10 targets both DNA and RNA.

DETECTION OF L. MONOCYTOGENES USING CRISPR/CAS SYSTEM
Listeria monocytogenes is one of the most virulent foodborne pathogens 43 and can be found in a variety of foods like milk, milk products, eggs, poultry, and meat. 44 The FDA upholds a zero-tolerance policy for L. monocytogenes since it has a low infectious dose and high mortality rate. In healthy people, it can cause invasive listeriosis. In young, elderly, or immunocompromised people, it can cause septicemia, meningitis, and infections related to the central nervous system. Infections in pregnant women can be fatal and can result in spontaneous abortion or fetal death. 45 The slow growth rate of L. monocytogenes is challenging for the conventional culture and plating-based detection methods, which can take up to 7 days to yield results. 46 CRISPR/Cas9-triggered isothermal exponential amplification reaction (CAS-EXPAR) based detection against Listeria monocytogenes was developed by Huang et al. 47 Here the hemolysin (hly) gene of L. monocytogenes was used as the target sequence. It utilizes the target-specific nicking activity of Cas9 and nicking endonuclease (NEase)-mediated amplification. From bacteria, RNA was isolated and cDNA was generated. cDNAs were cleaved by Cas9 with the help of specific sgRNA and PAMmers. These cleaved products are now subjected to EXPAR-mediated amplification by EXPAR templates and without exogenous primers. Finally, the amplified products were detected by fluorescence using SYBR green ( Figure 2a). This method combines the benefits of Cas9/sgRNA sitespecific cleavage and EXPAR fast amplification kinetics. This process is reported to be highly specific in discriminating single-nucleotide mismatch. Reprogrammable cleavage activity of Cas9/sgRNA is also beneficial for targeting various other pathogens. The merit of this method does not require exogenous primers for amplification. Therefore, the chances of nonspecific amplification followed by false positivity could be minimized. The sensitivity of this technique was reported to be 0.82 amol (Table 1) of synthetic ssDNA, but in bacteria, this technique was verified with 1.25 and 2.5 μg of total RNA. This method may have a problem to detect long targets, as EXPAR is not efficient for long DNA or RNA targets.
Another method to detect L. monocytogenes was developed by Wang et al. 48 based on CRISPR/Cas9 system integrated with lateral flow nuclic acid (CASLFA). Here also, the hly gene was used as a target. This technique is termed as the DNA unwinding-based hybridization assay with a lateral flow device for simple and easy detection by the naked eye. Genomic DNA from bacteria was subjected to amplification (by PCR or any isothermal reaction) with gene-specific biotinylated primers. Then biotinylated amplicons are incubated with target specific sgRNA and dCas9 to form a complex (Cas9/sgRNAbiotinylated amplicons) without cleaving the targets. This complex, when applied to a lateral flow device, bound with an AuNP-DNA probe (gold nanoparticle bound with complementary DNA sequence of target gene/sgRNA) and combined with immobilized streptavidin in the test line (T). Accumulation of AuNP-generated color band occurred on the test line (T). The excess unbound AuNP-DNA probe will form a control line (C) by binding with the precoated DNA probes with their control line hybridization region (Figure 2b). There are two kinds of DNA probes reported by the authors: DNA unwinding and sgRNA anchor-based. 48 The DNA unwinding probe has a specific DNA sequence for individual target. Therefore, for every target, there is a need to generate a new probe. The sgRNA-based probe has a target sequence that has DNA sequences specific to the crRNA region. The advantage of the sgRNA-based probe is that it can detect multiple targets as this is specific to a portion of sgRNA but not the target DNA. This is a simple and rapid method for the detection of genetic targets by the naked eye. This detection limit was reported as low as 150 copies (Table 1) of bacterial targets. As reported, this method can detect the gene target with almost no background signal interference and can be completed based on a cheap and portable tool kit within 40 min in point of care. 48

CRISPR/CAS-MEDIATED DETECTION OF M. TUBERCULOSIS
Tuberculosis (TB) is the leading cause of death among infectious diseases. 57 Due to the difficulty in its diagnosis, it was anticipated that 40% of the cases failed to be identified and reported. 50 Zhang et al. 49 have developed a novel and sensitive detection method to detect M. tuberculosis using CRISPR/dCas9. The Mtb 16S rRNA gene was used as a target sequence. In this method, the luciferase gene was split into N-and C-terminal halves (NFluc and CFluc) and fused each with separate dCas9 termed as a paired-dCas9 (PC) reporter system. Two guide RNAs (sgRNA) were also employed which are complementary to the upstream and downstream proximal segments (∼44bp) of a target DNA. Upstream and downstream sgRNAs are separately mixed with NFluc-Cas9 and CFluc-Cas9, respectively, to achieve higher efficiency and specificity. The two halves can initiate heterodimerization to rebuild the intact enzyme when target DNA containing the two segments in proximity is detected and bound by the corresponding sgRNAs followed by a pair of dCas9 (Figure 2c). Luminescence is generated from the catalytic activity of luciferase. The PC reporter system was reported to be sensitive up to 5 × 10 −5 nmol/mL (Table 1) but also observed that when the target concentration was beyond 6 × 10 −3 nmol/mL detection signals decreased due to inefficient pairing between two halves of luciferase. 49 This method requires two target sites, for separate sgRNAs, spanning approximately 44bp, which could limit the selection of target region for other pathogens.
The other method to detect M. tuberculosis by CRISPR-MTB was developed by Ai et al. 50 Here MTB-specific insertion sequence IS6110 of ∼1.5 kb in length was used as the target sequence. In this method, optimization of the extraction process was done based on a combination strategy of bead beating, chemical lysis, and heating to ensure the higher efficiency of DNA extraction. As the amplification technique is RPA, there was no special need for any thermal-cycler. This method is a combination of RPA reaction with a CRISPR/ Cas12a system (Figure 2d). Fluorescence signal can be detected by target activated reporter cleavage of Cas12a trans cleavage activity. The sensitivity of this method was reported to be 2−5 copies/μL or 50 CFU/mL (Table 1). It was reported that the extraction efficiency was found to be high and the extraction time was reduced with fewer centrifugation steps over the column-based traditional method. This method also was tested on a variety of sample types such as sputum, BALF, CSF, and pus. The drawback of this detection will be that it will fail to detect MTB strains that lack IS6110 genomic segments.  64 and pulsed-field gel electrophoresis (PFGE) 65 are labor intensive and require a high level of professional expertise. Therefore, MRSA detection requires simplified detection procedures that are faster, less labor-intensive, and highly specific. Kyeonghye Guk and colleagues recently introduced a CRISPR-mediated DNA-FISH. 51 This CRISPR-mediated DNA-FISH was developed to detect methicillin-resistant Staphylococcus aureus (MRSA) by targeting the gene mecA. This technique involves dCas9 to specifically recognize the target gene without cleavage activity and SYBR Green as a fluorescent probe. The genomic DNA of the target organism was isolated and treated with dCas9/sgRNA for 15 min at room temperature to bind the dCas9 with the target. After hybridization, the dCas9/sgRNA complex was isolated using Ni-NTA magnet beads, and nontarget unbound DNA was removed by washing. SYBR green was added to detect the presence of bound DNA as a target (Figure 2e). In clinical isolates, this method can detect as low as 10 CFU/mL within 30 min (Table 1). This approach is both quick and sensitive. This method does not require any amplification which is an advantage that reduces the detection time and complexity. The combination of dCas9/sgRNA and SYBR green as a fluorescent probe makes for labeling a reasonably straightforward and inexpensive approach. This method of detection has great potential to be used easily in patient point of care.

METHICILLIN-RESISTANT S. AUREUS (MRSA) DETECTION BY CRISPR/CAS SYSTEM
Suea-Ngam et al. have developed another amplification-free method for the detection of MRSA. 66 Here also, the mecA gene was chosen as the target. In this method of detection, the silver metallization technology was combined with the CRISPR/Cas to create a novel silver-enhanced E-CRISPR biosensor (E-Si-CRISPR) for MRSA detection. In the presence of target DNA the Cas12a−gRNA complex cleaves the ssDNA at random sites, destroying the electrode's ssDNA surface layer ( Figure  2f). The trans-cleavage mechanism fails in the absence of the target. The degree of silver deposition during the succeeding silver metallization stage is proportional to the quantity of ssDNA left and thus proportional to the initial amount of target DNA. Square wave voltammetry was used to read the final electrochemical signal (Figure 2f). The detection and quantification limits were found to be 3.5 and 10 fM (Table 1). This new electrochemical CRISPR/Cas biosensor, based on silver metallization, was stated to be highly selective, sensitive, and without DNA amplification cycles. As reported, this amplification-free detection method could yield results within 1.5 h. 66 This method is innovative in the aspect of its unique readout of results through electrochemical signals. One drawback can be perceived that, contrary to the other conventional methods, the positive signals are lower than the negative signals in this method, which could be inconvenient.

DETECTION OF SALMONELLA BY CRISPR/CAS SYSTEM
Food poisoning by Salmonella species is the second most prevalent cause of food poising followed by severe gastroenteritis and bacteremia worldwide. 67 To date, traditional biochemical culture, immunological testing, and molecular biological approaches (PCR/real-time PCR) have been used to detect Salmonella. 68 These procedures are time-consuming, have low specificity, and require expensive laboratory equipment. 69 Detection of Salmonella enteritidis using a unique allosteric probe (AP) with a combination of CRISPR/Cas13a (APC-Cas)is developed by Shen et al., 53 where whole bacteria were used as a target. The allosteric probe (AP) comprises of three functional domains (Figure 3a): aptamer domain for target pathogen identification (purple), primer binding site domain (blue), and T7 promoter domain (yellow). A phosphate group was added to the 3′ end of AP to prevent self-extension and make the DNA molecule resistant to enzymatic hydrolysis. The aptamer domain of AP can specifically recognize and engage with the target pathogen that is Salmonella enteritidis. The hairpin structure of AP will unfold and flip to its active configuration, allowing primers to anneal to the exposed primer binding site domain. The AP then acts as a template for the production of double-stranded DNA (dsDNA) with the help of DNA polymerase, followed by the displacement of the target pathogen for the next catalytic cycle (primary amplification) because of the polymerase extension reaction. T7 RNA polymerase is then utilized to identify the T7 promoter sequence on the created dsDNA and perform amplification via transcription to generate a large number of single-stranded RNAs (ssRNAs) (secondary amplification). Finally, the crRNA is intended to contain two areas, a guide sequence that is complementary to the transcripted ssRNA, and the repeat sequence that is required for crRNA to attach the Cas13a enzyme. When the above ssRNAs hybridize with Cas13a/crRNA, Cas13a/collateral crRNA's cleavage capacity is activated, allowing numerous RNA reporter probes to be cleaved (tertiary amplification), resulting in the amplification of fluorescence signals (Figure 3a). This procedure does not require bacterial isolation, nucleic acid extraction, and a washing step. It is cost-effective, very sensitive up to 1 CFU (Table 1), and can be done in a relatively short period. 53 Designing an allosteric probe for different bacteria could be a challenging task for this method of detection.
In order to detect Salmonella, Ma et al. 54 have developed gold-nanoparticles (AuNPs)-based method termed as CRISPR/Cas12a-powered dual-mode biosensor. The target DNA was Invasion gene A (invA), a virulence gene of Salmonella. 70 This method involves DNA extraction as well as PCR amplification of target sequence. The designed biosensor is built on the trans-cleavage activity of the CRISPR/Cas12a. The AuNPs probe is coated with DNA, and a linker ssDNA hybridizes with AuNPs-DNA probe pairs. In the absence of target amplicons, the linker ssDNA remains intact, and aggregated AuNPs are maintained with a purple color ( Figure  3b). Upon recognition of the target amplicons with designed crRNA, the trans-cleavage of CRISPR/Cas12a is activated and the linker ssDNA is cut off, and the AuNPs are dispersed in solution. The dispersed AuNP solution exhibits a red color, and the change can be detected by the naked eye or colorimetrically or photothermally (Figure 3b). The detection limit for this technique was reported as 1 CFU/mL (Table 1). This method was just used to detect the bacteria in milk samples. There is a need to explore with other food samples. This technique was the first to explore the gold-based nanoparticles as a probe. 54

CRISPR/CAS-MEDIATED DETECTION OF E. COLI
Escherichia coli O157:H7 is one of the most common causes of hemorrhagic colitis. 71,72 E. coli O157:H7 can be found in water as well as other food sources such as milk, juice, fruits, and vegetables. Infections that are severe enough can lead to hemorrhagic colitis, hemolytic uremic syndrome, and even death. 73,74 A CRISPR/Cas9 triggered SDA−RCA method on the UiO66 platform was developed by Sun et al. 55 to detect Escherichia coli O157:H7. The method employs the target sequence of gene hemolysin A (hlyA). Nanoparticle (UiO66) and Two amplification methods: strand displacement amplification (SDA) and rolling circle amplification (RCA) are used for this method. After isolation of DNA from the bacteria, the pair of CRISPR/Cas9 (by sgRNA1 and sgRNA2) recognized and cleaved the two proximal regions of the target DNA. Primary amplification by SDA synthesis and extending at the nicked position results in short−ssDNA indefinitely. This short−ssDNA was the template for secondary amplification by RCA, which generates long−ssDNA having repetitive sequences complementary to the fluorescence-labeled DNA probe (Figure 3c). This probe in bound form with long− ssDNA can be detected with 480 and 518 nm of excitation and emission wavelength. Unbound probes are absorbed in the UiO66 where fluorescence is quenched. In the presence of the target sequence, short (by SDA) followed by long ssDNA (by RCA) will be generated. The fluorescence probes will leave UiO66 and hybridize with the long−ssDNA, resulting in a fluorescence signal. As a result, the fluorescence intensity can be used to detect the target DNA quantitatively (Figure 3c). It is reported that this technique can detect low amounts of E. coli O157:H7 (40 CFU/mL) with high sensitivity and a wide detection range under mild response conditions (Table 1).

CRISPR/CAS-MEDIATED DETECTION OF PSEUDOMONAS AERUGINOSA
Pseudomonas aeruginosa is a multidrug-resistant, highly infectious opportunistic human pathogenic bacteria with a large and complex genome. 75 Its widespread distribution in nature indicates a high level of genetic and physiological flexibility in response to environmental changes. 76 In 2017, the World Health Organization designated P. aeruginosa as a critical pathogen that poses a serious threat to human health, necessitating the development of new treatments. 77 Mukama et al. have developed a method to detect P. aeruginosa 37 based on CRISPR/Cas and loop mediated Isothermal Amplification (CIA). The acyltransferase gene from P. aeruginosa was used as a target. Here the samples were directly used for loop-mediated isothermal amplification (LAMP) for the target gene. Products of LAMP were incubated with CRISPR/Cas12, to activate the collateral cleavage of the biotinylated ssDNA reporter, followed by a run on a strip for final results (Figure 3d). In the absence of a target, the gold nanoparticle-streptavidin (AuNP-SA) complex binds with the biotin of the ssDNA reporter. Then the whole complex (AuNP-SA-ssDNA) binds with complementary DNA to the ssDNA reporter immobilized in the test (T) line which results in a visible colored band (Figure 3d). But, in the presence of a target, the reporter DNA was cleaved by CRISPR/Cas12, so there will not be any formation of the AuNP-SA-ssDNA complex, hence no visible band on the test line. In the control line (C), only AuNP-SA is bound with immobilized antibodies to streptavidin. That means in the presence of any P. aeruginosa, there will be only one band in the control and no visible band in the test line ( Figure 3d). Whereas, if the sample is negative for P. aeruginosa then the visible band will be there in the T as well as in the C line in the strip. This method was reported to be a fast, accurate, robust, and inexpensive technique with a detection limit of 1 CFU/mL ( Table 1). The best feature of this approach is that it allows for naked-eye detection. That means this detection technique has the potential to apply in the patient point of care. On the other hand, the method of detection is unconventional too. In general, we are accustomed with a positive sample with a positive band in the test line, but here the positive sample is associated with a negative band in the test line, which may have some inconvenience in the hand of technicians. Additionally, we cannot eliminate the possibility of false positivity due to various reasons like degradation of ssDNA reporter, AuNP-SA, or complementary DNA in the test line.

CRISPR/CAS-MEDIATED DETECTION OF A. BAUMANNII
The rapid emergence of multidrug-resistant A. baumannii has posed a severe threat to worldwide public health. 78 In humans, it can be an opportunistic pathogen that affects immunocompromised persons and is becoming more common as a hospital-borne (nosocomial) infection. 79 Detection of A. baumannii based on the CRISPR/Cas system was developed by Wang et al. 56 This method was integrated with multiplex PCR where simultaneously many genes of β-lactamase, responsible for antibiotic resistance, were detected. Extracted genomic DNA from bacteria was used for the multiplex PCR reaction. When the target gene is present, the system will amplify the target and then initiate Cas12a's nonspecific ssDNA trans cleavage activity. The ssDNA reporter, conjugated with fluorophore and quencher, was cleaved after the Cas12a-crRNA-DNA assembly, resulting in an increase in fluorescence signals (Figure 3e). Different crRNAs were used to detect different genes. The detection limit was reported to be 50 CFU/mL ( Table 1). Integration of multiplex PCR provided an added advantage where multiple targets can be detected at once, provided the individual target specific crRNA needs to be developed.

DISCUSSION
All the methods discussed above based on CRISPR/Cas-based approaches for bacterial detection are distinct in their own way, employing various Cas enzymes and techniques. According to the ASSURED standards of WHO, all the above-described methods may not fully qualify all the standards, whereas some might be more affordable and others might be more sensitive or have less bulky equipment, or be easy to use in point of care. Likewise, amplification free methods, such as DNA-FISH 51 and E-Si-CRISPR, 66 need less time and fewer components, making them relatively inexpensive. Methods like CASLFA, 48 APC-Cas, 53 CIA, 37 and CRISPR-MTB 50 can also be conducted utilizing the costeffective isothermal amplification technique. 80 The CASLFA, 48 CIA, 37 and CRISPR/Cas12a-Powered Dual-Mode Biosensor 54 have naked eye readout capabilities requiring no expensive equipment. The APC-Cas 53 technique also does not need bacterial isolation or DNA extraction, which contributes to its low cost. The turnaround times for the methods described above ranged from 30 to 140 min. The CRISPR-mediated DNA-FISH for Methicillin-resistant Staphylococcus aureus (MRSA) detection 51 has the quickest turnaround time of 30 min as it is an amplification free method. In terms of the analytical sensitivity of the CRISPR/Cas assays, the limit of detection (LoD) was reported in different units: copies/mL, CFU/mL, moles, and molarity. The majority of methods reported the LoD in CFU/mL, with the lowest reported LoD being 1 CFU/mL for both CRISPR-Cas12a-powered dualmode biosensor 54 and CIA 37 (Table 1). APC-Cas 53 and CASLFA 48 was reported to be 1 CFU and 150 copies (Table  1). Between dCas9 49 and E-Si-CRISPR, 52 where both the LoD were reported in molar concentration, the lowest being for E-Si-CRISPR 52 that is up to 3.5 fM ( Table 1). The LoD of CAS-EXPAR 47 was estimated to 0.82 amol. Expressing the sensitivity with different units is misleading; therefore, it is hard to compare among methods. In the case of the bacterial detection method, it would have been helpful for the readers, if the sensitivity could have been reported to the standard unit that is CFU/mL. The CRISPR-based methods reduced the need for large equipment, which is a notable feature that has significant possibilities for field implementation, particularly for controlling epidemic outbreaks in resource-limited areas. CRISPR/ Cas systems make it easier to create a wide range of readout signals from fluorescence to naked eye detection. Methods based on LFA such as CASLFA 48 and CIA 37 might have higher utility at the patient point of care. Though CRISPR/Cas-based pathogen detection technologies have exceptional sensitivity and specificity, there are yet many scopes for future advancements. Due to the concerning inherent off-target impact of CRISPR-based detection, improved specificity is the utmost requirement for practical detection approaches. 36 For example, outside of the PAM-proximal 5−12 bp seed areas, Cas9-mediated cleavage is very tolerant to mismatches, 81 and dCas9 off-target binding is random, 82 which can weaken the analytical specificity and sensitivity. In the past few years, Cas9 variants with reduced off-target cleavage, such as SpCas9-HF1, 83 eSpCas9 (1.1), 84 and HypaCas9, 85 have been developed, thereby providing potential future solutions for the off-target effect of Cas9. Therefore, for Cas enzymes, future research should be concentrated on high-fidelity nucleic acid detection to minimize off-targeting. A more user-friendly one-step diagnostic that comprises pathogen nucleic acid release, preamplification, CRISPR/Cas-induced reaction, and signal readout should be developed in the future. For example, several simple formats, such as paper-based biosensors with visual readout (NASBACC, 86 SHERLOCK, 40 DETECTR 87 ), pathogen detection without nucleic acid extraction (HUD-SON), 88 and a single-tube test combining isothermal amplification and Cas-mediated reaction, have been developed, 39,89 which might be combined for more simplicity, affordability, and user-friendliness. More equipment-free approaches for signal readout, such as lateral flow assay 39 or naked-eye view 89 under light, should be introduced, as they might be easier at the point of care. Currently, the CRISPRbased detection system must be stored and delivered in a cold chain, which is an inconvenience in many remote areas. The NASBACC 38 detection system with freeze-dried reagents and the SHERLOCK 40 system with freeze-dried and paper spotting reagents showed long-term storage and transport. Therefore, more storage and transportation strategies for the CRISPRbased reaction kit should be developed.

CONCLUSION
CRISPR/Cas-mediated detection system is a very powerful and advanced technique with high specificity and sensitivity. Therefore, this CRISPR/Cas technique could be of high potential for early diagnosis in the present emerging scenario of antibiotic resistance. In most of the techniques discussed above for pathogenic bacterial detection, there are different amplification techniques like PCR, LAMP, RCA, and SDA integrated along with the CRISPR/Cas system. Previously, only positive amplification was sufficient for detection, but due to the high rate of false positivity, reliance toward only amplification to detect pathogens accurately becomes untrustworthy. Therefore, target amplification followed by CRISPR/Cas as biosensor-mediated detection of pathogenic bacteria can make the process more robust, reliable, sensitive, and specific.