EFFECT OF CRYOPROTECTANTS AND CRYOPRESERVATION METHODS ON THE QUALITY OF BUFFALO OOCYTES

This study was conducted to evaluate the effect of two types of cryoprotectants and two cryopreservation methods on viability and normal morphology of the immature and in vitro mature of post-thawed buffalo oocytes. The in vitro mature and immature oocytes were kept in the vitrification solution (VS1) for 5 min and the vitrification solution (VS2) for 15 sec. The results revealed that the proportion of buffalo oocytes found to be viable and normal morphology were significantly (P< 0.01) higher in using glycerol which is 76.14% and 55.1%; respectively. We conclude from the present study that the viability and normal morphology of post-thawed buffalo vitrified in vitro matured oocytes were significantly (P < 0.01) higher in glycerol than those obtained in dimethyl sulfoxide (DMSO) and in vitrification technique than rapid technique.


One of the most important challenges facing
Iraqi agriculture is protecting and improving national agricultural resources, including buffalo, to achieve sustainable food security (2).Buffaloes have low reproductive efficiency, which is affected by late maturity, poor expression of estrous symptoms particularly during summer, irregular estrous cycle, silent heat, poor pregnancy rate, early embryonic mortality, and prolonged intercalving interval (14).Reproductive traits are important in minimizing cost and maximizing the net return from the dairy enterprise (15).Reproductive efficiency is an important attribute even where most of the emphasis is on the milk (1).Genetic improvement of buffalo can be achieved by the application of assisted reproductive technologies, however, in buffaloes, those methods were not as efficient as in bovine (22).Cryopreservation of oocytes and embryos is the key to preserving genetic resources for future protection and managing species population integrity and heterozygosity (22), and it is helpful to enhance the better handling of buffalo gametes for both research and commercial purposes (3).Vitrification is a process that induces the glass-like solidification of living cells in high concentrations of cryoprotectant agents (CPAs) and rapid freezing at -196°C, minimizing the ice crystal formation that causes cryoinjury (14,19), The vitrification technique of oocytes may introduce a solution to low survival and low implantation rates problem in slow freezing method (6).Oocyte vitrification is now considered an established assisted reproductive technology for fertility preservation, (18).Rapid freezing (vapor phase) can be considered a midway technique between slow freezing and vitrification, it is quicker than the slow-freezing technique, does not involve the use of programmable machines, and requires lower concentrations of cryoprotectant agents (CPA) than those used in vitrification (28,10).A cryoprotectant is a substance used to protect biological tissue from freezing damage, is simply by increasing the total concentration of all solutes in the system, reducing the amount of ice formed at any given temperature; but to be biologically acceptable they must be able to penetrate the cells and have low toxicity and prevents lipid peroxidation of the plasma Membrane.(14,24).Many compounds act as cryoprotectants and are used for the protection of cells against freezing, there are two types of cryoprotectants: (a) Penetrating cryoprotectants are small molecules able to cross cell membranes, such as glycerol (G), ethylene glycol (EG), and dimethyl sulfoxide (DMSO); (b) Non-penetrating cryoprotectants were large molecules, such as Polyethylene glycol (PEG) and polyvinylpyrrolid-one (PVP) (13).To improve the effectiveness of vitrification and rapid freezing of buffalo oocytes, the present study evaluated the comparative effect of cryoprotectants (glycerol and dimethyl sulfoxide) on the viability and normal morphology of vitrified-thawed in vitro mature and immature buffalo oocytes.

MATERIALS AND METHODS
The buffalo ovaries from the normal reproductive tract of adult buffaloes of unknown breeding history were collected from a local abattoir in Al-Najaf Al-Ashraf Province from January 2021 to April 2021.Both ovaries were collected directly from each buffalo after being slaughtered and kept in a thermos flask containing warm normal saline (0.9% NaCl) inserted with antibiotics (100IU/mL penicillin and 100 μg/ml streptomycin).Ovaries were transported to a well-equipped laboratory Unit at the College of Medicine, Jabir Ibn Hayyan Medical University, Al-Najaf Al-Ashraf province within 2 hours.In the laboratory, ovaries were washed with warmed normal saline solution (37.5 C°) three times to remove the clotted blood and reduce contamination on the ovarian surfaces (6,21).

Cryopreservation of oocytes:
Normal and viable oocytes (immature and in vitro mature) were transferred to 0.5 mL of the vitrification solution (VS1) at room temperature to equilibrate for 5 minutes for oocytes.Then, oocytes were placed into 0.5 mL of vitrification solution 2 (VS2) for 15 seconds, then the oocytes were loaded on the straw of VS2 and directly immersed into LN2.Then, the straw is covered with the plastic tube in the LN2 to protect it during storage.The samples were pipetted into cryovials, the cryovials were left at room temperature for 10 minutes, and then frozen by static-phase vapor cooling and plunged into liquid nitrogen vapor (10 cm above the level of liquid nitrogen; -80 C°) for 15 minutes, The samples were then plunged into liquid nitrogen (-196C°) and stored until required, thawing was carried out at room temperature (9).

Thawing of oocytes
For thawing, the straws and cryovials were taken out from the LN2 after two months and immersed in the thawing solution at 37 ºC for 1 min, and then washed twice with SMART medium and tested for viability and morphology directly post-thawing.The thawed oocytes were considered abnormal when some changes occurred in shapes such as breakage of zona pellucida, uneven granulation, and leakage of oocyte contents (22).

Evaluation of oocytes
Livability was assessed according to El-Sokary et al. (10) using trypan blue stain solution (0.05%, PH=7.0 2min) at room temperature, hence, exclusion of stain by oocytes is indicative of viability.Oocytes were categorized based on the degree of dye exclusion, Unstained oocytes were classified as live and fully stained oocytes as dead (Plate 1)(4).

Plate 1. Viability and nonviable of buffalo oocyte (X=200). Statistical analysis
The statistical analysis system (SAS) program (26) was used to determine the effect of two types of cryoprotectants in post-thawed oocytes.The analysis of variation (ANOVA) and Duncan's Multiple Range Test for viability were used to compare between means.

RESULTS AND DISCUSSION
In this study, several buffalo oocytes were collected (427), All the oocytes were viable and had normal morphology, it's observed Highly significant (P<0.01)differences were assessed in the percentage of viability (%) and normal morphology (%) of vitro matured comparison with immature of post-thawed buffalo cryopreserved oocytes using glycerol, which showed that the best result is achieved in in vitro matured oocytes with using glycerol, which is 76.14% for viability and 55.18 % for normal morphology ( Table 1,2).The current study constructs that the viability and normal morphology of post-thawed buffalo cryopreserved oocytes decreased in samples vitrified with DMSO than those vitrified with glycerol in all treatments and this reflects the dominance of glycerol in the protection of vitrified oocytes.These findings compare favorably with Wani et al.( 29) illustrated that the high recovery rate of buffalo immature oocytes is cryopreserved of morphologically normal oocytes in all the concentrations of all cryoprotectants (Glycerol, EG, DMSO, and PROH).Also, Farshad et al. (11) showed when studying the effect of different concentrations of glycerol and DMSO on Makhoz goat spermatozoa, there was a significant difference in viability and motility by increasing glycerol concentration (P˂0.05).Furthermore, Rasul et al.( 25) which studied the effect of glycerol and DMSO during cryopreservation of buffalo semen, showed that within 6% glycerol treatment, post-thaw sperm is 47 ± 6.9% without DMSO, which reduced to 29 ± 3.1 and 21 ± 3.1% in the presence of 1.5 and 3% DMSO, respectively (P < 0.05).Likewise, post-thaw plasma membrane integrity was the highest in 6% glycerol without DMSO (47 %) than with DMSO (32 %, P<0.05).Glycerol demonstrated a strong affinity with the cells' phospholipid head groups during freezing (5).The cryoprotection of glycerol is due to its ability to buffer the salt at low temperatures, bind with metallic ions, dehydrate the cell, and reduce the ice expansion during water solidification (7,17).The absence of glycerol in the extender led to a complete cessation of sperm movement, despite negligible protection to the plasma membrane and acrosome (25).Furthermore, glycerol acts by reducing the concentration of the electrolyte in the extracellular fluid and increasing the portion of the unfrozen solution at any temperature (16).In addition to its effect on preventing the formation of ice crystals and the dehydration of cells during the cryopreservation process (30).Results of the present study indicated a too-high percentage of viability (78.11 %) and normal morphology (58.14%) of post-thawed buffalo cryopreserved oocytes when using the vitrification technique and glycerol, but there were non-significantly (P˃0.05)differences were assessed when using a rapid technique with glycerol or DMSO on viability characteristic, which is 67.38% and 64.90%; respectively.This result is agreed with Mahmoud et

Table 2 . Effect of the relationship between the types of cryoprotectant with the type of oocytes on the percentage of normal morphology of post-thawed buffalo vitrified oocytes IVM: in vitro matured, DMSO: Dimethyl sulfoxide. Different letters in the table refer to significant (P < 0.01) differences.
IVM: in vitro matured, DMSO: Dimethyl sulfoxide.Different letters in the table refer to significant (P < 0.01) differences

Table 3 . The effect of the relationship between the type of cryoprotectants, and oocyte on the percentage of viability of post-thawed buffalo vitrified oocytes Type of Cryoprotectants Type of Cryopreservation Rapid tech.
DMSO: Dimethyl sulfoxide.Different letters in the table refer to significant (P < 0.01) differences

Table 5 . The effect of the relationship between the type of cryoprotectants, cryopreservation, and oocyte on the percentage of viability of post-thawed buffalo cryopreserved oocytes Type of Cryoprotectants Type of Cryopreservation
IVM: in-vitro matured, DMSO: Dimethyl sulfoxide.Different letters in the table refer to significant differences.** (P < 0.01).

Table 6 . The effect of the relationship between the type of cryoprotectants, cryopreservation, and oocyte on the percentage of normal morphology of post-thawed buffalo cryopreserved oocytes IVM: in-vitro matured, DMSO: Dimethyl sulfoxide. Different letters in the table refer to significant differences.
** (P < 0.01).