Effect of Polyvinylpyrrolidone on Vitrification of Buffalo ( Bubalus bubalis ) Oocytes

: Vitrification, a method of rapid cooling, is an alternate cryopreservation method of oocytes and embryos. The present study was aimed to examine the effect of polyvinylpyrrolidone (PVP) on vitrification of buffalo oocytes. Cumulus oocyte complexes (COCs) with fully grown oocytes (120-130 µ m in diameter) were aspirated from slaughtered buffalo ovaries for vitrification. COCs were treated with equilibration solution at room temperature for 5 min and then transferred to a vitrification solution for 1 min. Then the COCs were submerged into liquid nitrogen (-196 ̊ C) for a while using cryotops. The COCs were thawed, diluted, and washed in a washing solution for 5 min, respectively. Vitrified oocytes were incubated for in vitro maturation (IVM) at 38.5 ̊ C under an atmosphere of 5% CO 2 in the air for 24 hrs. Cumulus cells surrounding the oocytes were removed mechanically, oocytes were fixed in acetic acid and ethanol, and stained with aceto-orcein to examine the meiotic stages of oocytes. The numbers of morphologically normal oocytes after vitrification were higher in 5% PVP than 0 and 10% PVP groups. A proportion of oocytes treated with 5% PVP reached the metaphase II (MII) stage while none of the oocytes from 0% and 10% PVP groupsdeveloped beyond anaphase I and metaphase I (MI) stages, respectively. These results show that PVP can be used as a cryoprotectant for the vitrification of that supplementation of vitrified-warmed ovarian


INTRODUCTION
The buffalo (Bubalus bubalis, L.) is an important livestock species in the world. Buffalo farming is highly emphasized for household income from milk for farmers' livelihood in Bangladesh [1]. Buffaloes have low reproductive performances caused by silent estrus, seasonal anestrous, delayed puberty, late post-partum conception, and a long calving interval. Buffalo oocytes collected from large antral follicles during the luteal phase are less competent due to the presence of different fatty acids in follicular fluid [2]. Due to low response to multiple ovulation and embryo transfer (MOET), in vitro embryo production (IVEP) technology is important in buffaloes. The major limitation of IVEP technology in buffalo is the small number of oocytes that can be recovered from donors. Buffalo ovary contains small numbers of antral follicles and a high percentage of atretic follicles [3]. It has been reported that buffalo ovaries have a smaller number of recruitable follicles [4] and lower reproductive hormones [5] at a given time compared to a cow. The scarcity of oocytes is a drawback for exploiting embryo technologies in buffaloes. Therefore, vitrification can be a useful technique to avail buffalo oocytes for reproductive technology.
Vitrification, a method of rapid cooling, is the solidification of a solution at low temperatures (-196 C) without ice crystal formation in the cell cytoplasm by use of a very high concentration cryoprotectants [6]. To improve the reproductive performance of water buffaloes, a method has been developed by Abdel-Ghani et al. for the in vitro culture of vitrified-warmed ovarian tissues [7]. They found that supplementation of vitrified-warmed ovarian tissue culture medium with growth differentiation factor-9 (GDF-9) promoted primary follicle development that might be used for improving ovarian inactivity in water buffaloes. Oocytes were collected from slaughtered water buffaloes, matured and fertilized in vitro, and embryos were cultured in cumulus cells monolayer, then resultant preimplantation stage embryos were cryopreserved and transferred to recipient buffaloes [8]. Six healthy and normal calves produced from that cryopreserved technologies indicated the potential application of vitrification techniques in buffalo reproduction regime.
In the year 2005, in vitro fertilized and cryopreserved water buffalo embryos were transported from Italy to California for transferring them into recipient buffaloes [9]. In the vitrification method, a cryo-device is required to hold the sample materials and be used for submerging into liquid nitrogen. A comparative study on cryo-devices has been done by Mahesh et al. [10]. They collected COCs from buffaloes and vitrified using either a conventional straw, open pulled straw, cryoloop, hemistraw or cryotop. Their results suggest that hemistraw, cryotop, and cryoloop are more suitable as cryo-device for vitrification of buffalo oocytes.
Vitrification requires the use of cryoprotectant solutions, which prevent ice crystal formation and increase viscosity at low temperatures. Several cryoprotectants, including ethylene glycol (EG), glycerol (GLY), dimethylsulfoxide (DMSO), propylene glycol (PG), and 1,2-propanediol (PROH), sucrose, etc. have been used in different combinations for vitrification of buffalo oocytes and embryos [11]. EG is an important cryoprotectant due to its higher penetrating ability with low toxicity [12]. EG and PG are equally effective in preventing cryodamage of buffalo oocytes [13]. Disaccharides are recently being used for the vitrification of oocytes in various species, including mouse [14], cattle [15], buffalo [16], and human [17,18]. Effects of different sugars (i.e. glucose, sucrose, or a polysaccharide) as non-permeant cryoprotectants in vitrification media on in vitro maturation of vitrifiedwarmed immature (GV) porcine oocytes have been studied by Huang et al. [19]. They reported that sucrose treated oocytes had a higher maturation rate compared to oocytes vitrified in glucose supplemented cryoprotectant.
In principle, short exposure to a high concentration of cryoprotectants has been widely used to reduce cell injury. In contrast, Mukaida (2002) has reported a high survival rate of blastocysts vitrified with a combination of EG and DMSO [20]. The combination of EG and DMSO for the vitrification process provides a lower relative concentration and also causes lower toxicity. In fact, when DMSO penetrates the cell and combines with other cryoprotectants, it accelerates glass-forming and increases the permeability rate complementing each other. DMSO has been used for the vitrification of oocytes in many species, including buffalo [21] and cattle [22]. However, it has been reported that DMSO adversely affects the developmental processes of oocytes [23].
Polyvinylpyrrolidone (PVP), also known as povidone, is a water-soluble polymer of N-vinyl-2pyrrolidone [24]. It has been used widely in pharmaceuticals, cosmetics, and food industries. It is used as a vehicle for drugs and a food additive. PVP has been used for intra-cytoplasmic sperm injection (ICSI) to increase the viscosity of sperm solution for easy handling of individual sperm in domestic animals and humans [25][26][27][28][29][30]. PVP prevents the adherence of oocytes to plastic and glass dishes. It has also been used in media for the in vitro growth of bovine oocytes [31]. PVP was used as a substitute for a serum for in vitro maturation of bovine [32]. Supplementation of PVP in EG and sucrose based vitrification solution resulted in an increased survival rate of bovine oocytes after vitrification and warming [33]. PVP is a nonpermeating cryoprotectant. It creates a smear around cells during vitrification and protects them from the cryodamage. It was reported that PVP protects the disruption of the zona of oocytes [34]. Supplementation of PVP in EG based vitrification media increased survivability of mouse oocytes [35]. PVP has a protective role in vitrification for survival and subsequent development of bovine oocytes [33]. However, so far, a few studies have been conducted to know the effect of PVP as a cryoprotectant for the vitrification of buffalo oocytes. This present study was aimed to examine the effect of replacing DMSO with PVP on vitrification solution for cryopreservation of buffalo oocytes.

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

Collection of Cumulus Oocyte Complexes (COCs)
Buffalo ovaries were collected from a local slaughterhouse and transported to the laboratory in 0.9% normal saline. The ovaries were washed in Dulbecco's phosphate buffer saline (DPBS) solution supplemented with gentamycin sulfate (50 mg/mL) once and rinsed three times in DPBS. Visible antral follicles (4-6 mm in diameter) were aspirated using a 20-gauge needle attached to a 10 ml syringe to collect COCs. The COCs were screened under a stereomicroscope and washed three times in TCM-199 (pH 7.4, Nissui Pharmaceutical, Tokyo, Japan) containing 0.85 mg/mL NaHCO 3 , 0.08 mg/mL gentamycin sulfate and 25 mM HEPES in a plastic dish (No. 1008, Falcon, Becton Dickinson and Company, Franklin lakes, NJ, USA) for vitrification. COCs containing healthy oocytes (120-130 µm in diameters) were selected based on their morphological appearances (uniformly granulated cytoplasm surrounded by multilayered compact cumulus cells) for vitrification [36].
At first, COCs were treated with equilibration solution at room temperature for 5 min and transferred to a vitrification solution for 1 min. Then, 3-5 COCs were loaded on the filmstrip of a cryotop (Kitazato Biopharma, Shizuoka, Japan). The surrounding solution was removed and immediately plunged into liquid nitrogen (-196C). After vitrification, the COCs were warmed in the thawing solution for 5 min. Then the COCs were placed in a dilution solution for 5 min. The COCs were washed in a washing solution for 5 min to remove the cryoprotectants. The COCs were evaluated for morphological quality as described by Gupta et al. with some modifications [36]. Briefly, COCs with multilayered compact cumulus cells and good integrity between oolemma and zona pellucida were classified as morphologically normal COCs. The COCs with normal morphology were subsequently used for in vitro maturation, and the rest were discarded from further experiments.

In Vitro Maturation (IVM)
The basic medium for oocyte maturation was TCM-199 supplemented with 0.1 mg/ml sodium pyruvate, 0.08 mg/ml gentamycin sulfate, 5% (v/v) FBS and 100 ng/ml follicle-stimulating hormone (FSH; NIDDK, Washington, DC, USA) [39]. The vitrified and thawed COCs were washed three times in the IVM medium. COCs were placed in 100 µL droplet of IVM medium in 35 mm Petri dish under mineral oil, and incubated at 38.5C, 5% CO 2 in humidified air for 24 hrs. After 24 hrs, oocytes were observed under the microscope for cumulus expansion. The assessment of cumulus cell expansion was carried out as described by Maruska et al. with some modifications [40]. Briefly, COCs with one or two layers expanded, one-half of the cumulus expanded, all layers expanded other than last layers of corona radiata, or all layers expanded, including corona radiate, were classified as expanded COCs. All of the COCs other than expanded COCs, such as COCs without cumulus expansion (no observable sign of cumulus expansion), were classified as non-expanded COCs. The oocytes were picked up from the droplets and washed in Dulbecco's phosphate-buffered saline (DPBS). The oocytes were denuded mechanically using a small-bore pipette with the help of 0.1% (w/v) hyaluronidase. Oocytes were fixed in acid-alcohol (acetic acid: ethanol = 1:3) for two days, stained with aceto-glycerol (glycerol: acetic acid: water = 1:1:3) and examined under a differential interference contrast (DIC) microscope (Olympus Inc., USA) for meiotic stages [39]. Oocytes were classified based on their chromosomal configuration following the previous report of Motlik et al. [41]. Oocytes showing cytoplasmic or nuclear abnormalities were considered degenerated.

Statistical Analysis
All data were subjected to one-way ANOVA, and the significance of difference among means was determined by Duncan's Multiple Range Test (DMRT). All statistical analyses were conducted using SPSS (IBM SPSS Statistics 22) software for Windows. Values of P < 0.05 were considered significant.

Effects of PVP on the Morphology of Vitrified Buffalo Oocytes
The numbers of oocytes recovered after vitrification did not differ among the treatment groups (Table 1). However, the number of morphologically normal oocytes was significantly (P<0.05) higher in 5% PVP than that of other groups. In a higher concentration (10%) and without PVP oocytes underwent to various abnormal morphological changes, e.g., shrinkage of oocytes cytoplasm, denudation of oocytes and dissociation of cumulus cells, etc.

Effects of PVP on In Vitro Maturation of Vitrified Buffalo Oocytes
A proportion of vitrified oocytes treated with 5% PVP reached the MII stage while none of oocytes in 0or10% PVP group progressed beyond Anaphase I and MI stage, respectively ( Table 1). The percentage of oocytes at the MI stage was higher in 5% PVP (40%) than in other groups. The cumulus expansion in 5% PVP treated oocytes were also comparable with that in control group, although there were no significant differences among the PVP treated groups (Table 2, Figure 1).

DISCUSSION
The present study showed that 5% PVP treated vitrified oocytes reached the MII stage while oocytes in other groups (0% or 10% PVP) resumed meiosis but did not reached the MII stage. PVP is a macromolecule that prevents oocytes from cryoinjury. It has been added to cryopreservation solutions for embryos in many species [42]. Yang     oocytes [33]. Among the treatment groups, the survival rate of oocytes was higher in 5% PVP than in 0% or 10% PVP. The rate of in vitro maturation was more elevated in 5% than the 10% PVP group. In the slow freezing of bovine oocytes, 5% PVP had a beneficial effect compared with 10 or 20% PVP [43]. On the other hand, Checura and Seidel (2007) reported the successful vitrification of bovine oocytes with 20 and 6% PVP [44]. Mouse embryo was successfully vitrified with supplementation of EG and sucrose as a cryoprotectant solution with 7.5% PVP [45].
Despite the protective effects, cryoprotectants impose toxicity to cells [45]. DMSO affects the organisation of the microtubule systems in mouse oocytes [23]. Cytoskeletal elements are implicated during the developmental process in oocytes [47]. It was reported that DMSO altered the microtubule organization of mouse oocytes [48]. DMSO caused progressive disassembly of the spindle that was demonstrated to be sensitive in chilling [49,50]. It lowered the critical protein content for microtubule assembly [51,52]. This may be the reason for the lower rate of meiotic progression in DMSO treated oocytes in this experiment. The present study showed that PVP could be a better replacer of DMSO in vitrification of buffalo oocytes. Previously it has been reported that PVP prevents zona pellucida of oocytes from cryodamage [53]. However, comparing the results in DMSO, PVP could not rescue buffalo oocytes from cryodamage as it was expected. In agreement with our results, it was reported that normal developmental competence of buffalo oocytes was compromised by vitrification with PVP [54].
PVP at a concentration of 5% increased morphologically normal COCs after vitrification than 0% and 10% of PVP. This indicated that PVP increased the survivability of oocytes during vitrification. PVP in the vitrification solution increased the survival rate of mouse oocytes after vitrification-warming [35]. Whittingham (1971) reported fruitful use of PVP for the cryopreservation of mouse 8 cell stage embryos and further early blastocysts [55]. When embryos were frozen at a temperature of -79°C in a 7.5% PVP solution, the survival rate was 55-65%. Damage to the zona pellucida after freezing and thawing in mouse embryos was accompanied by low survival rates of the embryo itself [56]. Since the survivability of oocytes and embryos during vitrification is largely depends on the zona pellucida and in this experiment, zona pellucida were examined to assess the morphologically normal oocytes. It is reasonable to conclude that PVP maintains the integrity of oolemma and zona pellucida, and cumulus cells and zona pellucida thereby increased morphologically normal COCs after vitrification.
It is thought that PVP is deleterious for the development of vitrified embryo [57]. It is toxic to kidney cells [45]. In the present study, a lower rate of meiotic progression was found in 10% PVP treated oocytes than in 5% PVP group. This might be due to the deleterious effect of a higher concentration of PVP on oocyte maturation. This supports the previous report of Wang et al. (2013), where mouse oocytes were vitrified with 2% PVP combined with other cryoprotectants [35]. However, further study is necessary to elucidate the mechanisms involved in the deleterious effect of a higher concentration of PVP on oocytes and embryos.
In conclusion, PVP protects buffalo oocytes from cryoinjury and supports the meiotic progression of oocytes in vitro after vitrification and warming. PVP at a concentration of 5% maintains the normal morphology and promotes meiotic maturation of oocytes after vitrification and warming, whereas 10% PVP exerts the toxic effects. Therefore, 5% of PVP could be used as a cryoprotectant for the vitrification of buffalo oocytes.

CONFLICT OF INTEREST
The authors declare that no conflict of interest could be perceived as prejudicing the research submitted impartiality.