The Impact of Follicular Fluid on the in vitro Maturation of Camel Oocytes

Many issues, maybe caused by insufficient final maturation, restrict embryo development in vitro. With respect to oocyte, a slow pace of embryonic development in vitro, So, it’s safe to say that in vitro maturation has a significant impact on the success of embryo evolution. According to the study conducted by Wani and Nowshari in (2005), it was determined that eggs need to undergo a maturation process in the laboratory for a period of 32-44 hours in order to achieve the highest number of mature eggs in the MII stage, which ranges from 42-52%.
       
The in vitro maturation rate (IVM) is the only one of the three major processes that are involved in the in vitro embryo production (IVEP) technology. This rate is rather low when compared to the pace at which oocyte mature in vivo.
       
Lu et al., (2018), improving the microenvironment of the oocyte such that it is comparable to that of the in vivo counterparts is the most difficult obstacle that IVEP must overcome. On the other hand, certain IVM components and conditions can be necessary (Watson et al., 2000; Barakat et al., 2015; Barakat et al., 2016; Al-Mutary et al., 2019). have all found that there are certain variations in the gene expression of oocytes that result in a decline in the quality of the oocytes. 
       
Various chemicals have been added to enhance the culture conditions of the in vitro maturation (IVM) process. One of these substances is the follicular fluid (FF), which contains a variety of nutrients that might potentially impact the development of the oocyte growth, fertilization and embryo development (Germoush et al., 2018).
       
FF has been utilized in intravenous methanol (IVM) studies in a variety of animals and plants, including cattle (Ali et al., 2004), goats (Masudul et al., 2012), pigs (Ito et al., 2008), buffalo (Gupta et al., 2005), horses (Bogh et al., 2002) and sheep (Sun et al., 1994). Nonetheless, oocyte and blastocyst gene expression may be affected by adding FF to in vitro maturation medium, which in turn affects the amount of messenger RNA (Watson et al., 2000; Cruz et al., 2014).
       
However, the influence of camel follicular fluid (CFF) supplementation during the in vitro maturation of camel oocytes on maturation has not been examined. In general, the competence of an oocyte is measured by the maturation rate, the fertilization rate, the cleavage rate, and the blastocyst ratio (Ammari et al., 2022a). Within the scope of this study, we investigated the impact that CFF has on oocyte maturation.

An aspiration was used to obtain the follicular fluid from intact follicles that were discovered in camel ovaries that had been taken from animals that had been slaughtered a slaughterhouse more recently. The fluid was collected in 15 ml centrifuge tubes and subjected to two rounds of centrifugation at 4000 rpm for 20 minutes each to eliminate cellular debris. The liquid portion was collected, passed through a 0.45 mm filter and subsequently rendered inactive by heating at 56°C for 30 minutes. It was then kept in separate portions in 1.5 ml Eppendorf tubes at a temperature of -20°C until needed (Al-Mutary et al., 2019).
       
Following oocyte collected from the ovaries, (COCs) were separated into two groups, with the distinction between the two groups being determined by the amount of camel follicular fluid (CFF) that was added to IVM medium: The control group had 0% CFF, 2.5% CFF and 5% CFF. Repeated pipetting was used to remove the cumulus cells that were surrounding the oocytes after they had matured. The oocytes were then placed in Hank’s medium that had been supplemented with 100 IU/m of hyaluronidase.
       
A local slaughterhouse located south of the city of Riyadh was be used to collect ovaries from adult females who have recently been slaughtered. These ovaries were then be brought to the laboratory in a container that contains 0.9% normal saline and was be kept at room temperature normal according to Ammari et al., (2022b).
       
The ovaries washed twice with a salt solution once they get to the lab. Then, the method called the Aspiration Method used to remove the oocyte from the main follicles, which were 2 to 8 mm in diameter. 10 ml syringe was connected to 19 diameter needles. Just before extraction, the oocyte collection solution, Hank’s Salt’s TCM-199, which is about half a milliliter in volume, was added to the syringe. The contents of the syringe will be carefully placed into a 15 ml conical tube after the drawing operation. After that, the oocyte was allowed to settle to the bottom of the tube by leaving it in the solution for 5 minutes. We remove the fluids. Then, use a clean 90 mm petri dish to transfer the remaining material containing the COCs. Next, gently we transferred the COCs (multi-layered with homogeneous cytoplasm) to the Petri plate containing the oocyte collection medium using a glass pipette. Utilizing TCM-199 in conjunction with glutamine, 25 mM of 4-(2-hydroxyethyl), and Hank’s salts forms the handling media. A -1 piperazineethanesulfonic acid (HEPES) supplement, 10% fetal calf serum, 0.3 mM sodium pyruvate, 25 mg/mL gentamicin sulfate and 143 mg/mL heparin sodium salt are all components of the prepared solution according to Ammari et al., (2023). 
       
The oocyte samples from each group, which had been stripped of their outer layer, were placed on glass slides. The slides were then coated and secured with a mixture of acetic acid and ethanol in a ratio of three parts acetic acid to one-part ethanol (v/v). This fixation process lasted for twenty-four hours and then stained with 1% aceto-orcein stain. Examples of the samples were taken from each group. An examination of the oocytes was carried out with the assistance of a phase contrast microscope (400x Olympus, CKX41 Japan). The nuclear state of the oocytes was evaluated, which included the presence of germinal vesicles, germinal vesicle breakdown, metaphase I and metaphase II (Fig 1).
 

       
The statistical analysis of the data involved using the SPSS version 20 software program. A minimum of three duplicate trials were conducted for each treatment comparison. The analysis was performed by SPSS Inc., based in Chicago, IL, USA. To begin the data analysis process, we utilized the Kolmogorov-Smirnov test to normalize the data. The means of GV, GVBD, MI, MII and degenerated oocytes throughout in vitro maturation were compared across all groups using one-way analysis of variance (ANOVA) and post-LSD Dunnett’s test.       

The results of our study indicate that adding follicular fluid to the maturation medium did not cause clear significant differences. This may be the reason for the decrease in the concentrations used, but on the contrary, the cumulus cells showed a clear expansion compare with the control group, which gives an indication that there is maturation at the cytoplasmic level, which encourages further studies and the use of higher concentrations may improve the maturation rates of camel oocyte.