Production of Parthenogenetic Blastocysts from Oocytes Aspirated of Ovine Ovaries with or without Corpus Luteum

Parthenogenetic activation

Activation dishes were prepared three hours before use. An aliquot of Di-Methyl-Amino-Purine (DMAP) was placed on the flame until it dissolved. 100mm dishes containing 50μL/ 25 zygotes were prepared using activation or culture medium. These are culture drops. The drops were covered with mineral oil and the plate was placed in the incubator with 5% CO₂ and 38.5 °C.

Three drops of oocyte transport medium (SOF-HEPES) were placed, and the oocytes were washed through each drop three times. The zygotes were transferred to 0.6mL of hyaluronidase in a microcentrifuge tube and the volume of hyaluronidase was lowered to 100μl, verifying that COC (cumulus-oocyte complex) had not been aspirated.

Subsequently, the microcentrifuge tube was placed in the vortex for 5 minutes and withdrawn every minute in order to remove the cells from the cumulus oophorus. 1mL of SOF-HEPES was added to the tube, the oocytes were mixed and removed, checking that there were no oocytes in the tube. The number of oocytes to activate was searched and a drop of SOF-HEPES was placed in which they were washed three times.

Then a 1.5mL tube was used, 1μl of ionomycin and 1mL of SOF-HEPES were added and mixed. The oocytes were placed in 1 drop of ionomycin/SOF-HEPES mixture and allowed to stand for four minutes in total darkness. After this time, the oocytes were washed three times in drops of SOF-HEPES and once in BO-IVC + DMAP, they were separated into groups in BO-IVC + DMAP dishes. They were incubated on a plate with KSOM + DMAP medium for 4 hours. After the elapsed time, the oocytes were washed again in 3 drops of SOF-HEPES and once in BO-IVC + BSA.

In vitro embryo culture

The culture medium was prepared three hours before use. 60mm Petri dishes containing 50μl drops of culture medium/25 zygotes were prepared, covered with mineral oil and then the dishes were placed in the incubator with 5% CO₂, 38.5 °C and relative humidity saturation. The zygotes were transferred to a culture plate containing 50µl of mineral oil coated culture medium. The dish was placed in a chamber and the gas inside the chamber was replaced with an air mixture of 5% O₂, 5% CO₂ and 90% N₂ for two minutes and then placed back in the incubator at 5% CO₂ and 38.5 °C. On day three of the culture, it was supplemented with 2.5μl of Fetal Bovine Serum/drop of culture (Gemini Bio 100-525). On day 7, blastocyst formation was evaluated.

Treatments

Three treatments were developed
1) oocytes from sheep ovaries in the anestrus state (without corpus luteum)
2) oocytes from sheep ovaries with corpus luteum present.
3) oocytes from ovaries of pregnant ewes.

Variables analyzed

Average of oocytes collected per ovary, percentage of viability of the oocytes according to the number of layers of cumulus oophorus cells and the homogeneity of the cytoplasm, percentage of cleavage at 72 hours, percentage of parthenogenetic blastocysts and general efficiency.

Experimental design and statistical analysis

A Completely Random Design was used with three treatments and two repetitions per treatment. The percentage values were analyzed using the Chi-square (χ²) frequency distribution test; For the numerical values of extracted oocytes and number of oocytes/ovary, an Analysis of Variance (ANOVA) was used applying the General Linear Model (GLM) and Duncan's multiple range test, with a required significance value. of P≤0.05, using the "Statistical Analysis Systems" program [4].

Oocytes collected per ovary

The differences were significant (P=0.0001) between the treatments, being the treatment of the aspirated ovaries with corpus luteum the one that obtained the highest number of aspirated oocytes/ovary with a value of 3.33 followed by the ovaries of pregnant females with 1.52 and the smallest for ovaries without corpus luteum with 1.24. These results are attributed to the fact that by presenting the ovary the corpus luteum has as its main action the production of the hormone progesterone, which has an effect on follicular development since it is responsible for inhibiting the dominant follicle, therefore, by generating the Regression of the dominant follicle creates a new wave of follicular development [5]. Sheep have one or two ovulations per cycle and this variation depends on genetics, age, season, and nutrition. In this study, ovaries were taken from a slaughterhouse where the age of the sheep is unknown, but it is known that they were taken to the slaughterhouse during the short days that was the month of January, where they show greater cyclicity and prolificacy.

Percentage of viability

There were significant differences (P≤0.05) between the treatments (Table 1), being the treatment of the aspirated ovaries with corpus luteum the one that obtained the highest number of viable oocytes, surpassing the treatments of ovaries without corpus luteum and ovaries of pregnant females in 54.13% and 43.2% viability respectively. The viability of oocytes is related to the ability of oocytes to generate an embryo, ovaries with corpus luteum have a high percentage of viability. The higher the circulating progesterone, the concentrations promote follicular turnover and with the new wave of ovarian follicular development, it results in a viable oocyte that is released [6]. According to studies carried out by Menchaca et al. [6] mention that by affecting the competition of the oocytes with progesterone, they generated viable embryos. What is attributed to the fact that the results of the present investigation when comparing the treatments, ovaries with corpus luteum have given greater results than the other treatments.

In the present investigation, oocytes with many compact layers of cumulus oophorus cells and homogeneous cytoplasm were taken, which is an indicator of high quality oocytes, that is, they will be viable for the production of embryos in vitro and according to a study carried out by Córdova et al. [7] follicles larger than 3mm have more layers of cumulus, which generates better maturation in vitro, being an important aspect to take into account when selecting oocytes at the end of their growth phase. This is attributed to the fact that the oocytes will have nutrients and energy to be able to develop better compared to others that are grade C and D, since these oocytes are discarded because they do not have enough layers of cumulus oophorus and are not suitable for use in embryo production.

According to Lonergan [8], the quality of the oocyte is based on different aspects such as the physiological and reproductive state of the donor, the follicular size and the amplitude and integrity of the cumulus cells. In this study the breadth and integrity of the cumulus cells is known, and these played an important role for the maturation of the oocytes since they provide energy and nutrients to the oocyte, in this study oocytes with high quality cumulus layers were selected along it is attributed that the results have provided a percentage greater than 50%.

The morphology of the aspirated oocytes from the ovaries allows the possibility of predicting their ability to restart meiosis [9]. When analyzing the three treatments, it is observed that the oocytes from the ovaries with corpus luteum stood out since at the time of selecting and classifying them all were viable, these results are attributed to the cyclical activity that these sheep presented in vivo, since they were in the riding season which is characterized by short days resulting in greater cyclicality.

Cleavage percentage

The differences found were not significant (P>0.05) between the treatments (Table 1), with all treatments exceeding 50% cleavage. These results are good because in all treatments they presented cleavage greater than 50%, which may mean better results for the formation of a viable blastocyst.

The cleavage of oocytes is important since it means that when the cells are dividing, the creation of blastocyst can be achieved [5]. This study sought to analyze the production of blastocysts by parthenogenetic activation that are linked to the cleavage of oocytes. When analyzing the results, it was determined that the oocytes independent of the treatment presented a cleavage of more than 50%, but an effect was not obtained that differentiates whether a treatment is significant compared to the others, so it is inferred that the selection and classification of the oocytes play an important role in the competition of oocytes and thus in obtaining acceptable cleavage rates. Although the treatment of oocytes from ovaries with corpus luteum showed the highest viability, this effect was neither reflected nor maintained in cleavage.

Percentage of parthenogenetic blastocysts

There were no significant differences (P>0.05) between the treatments (Table 1), in general, all treatments exceeding 35% of parthenogenetic blastocyst production. According to studies carried out by Menchaca et al. [6] blastocyst production trends have been established, with 50% of the oocytes generating blastocysts. When comparing with the present study, lower results (27.85%) were obtained than those obtained by this author.

Table 1:Mean values of viable oocytes, cleavage, and blastocyst rate of sheep, according to the physiological condition of ovaries with corpus luteum, without corpus luteum and pregnant.

^abAverage values in columns with different letters, differ statistically from each other (P≤0.05).

In vivo cultured, fertilized oocytes yield between 60 and 70% blastocysts, but when in vitro matured blastocysts are used, the development rate is 50%, according to a study by Córdova et al. [7]. If the objective is to generate the greatest number of blastocysts, it is necessary to select competent oocytes for in vitro maturation, fertilization and development, fulfilling this condition only grade A and B oocytes, however, this varies greatly depending on genetics and ambient.

The embryos produced by parthenogenetic activation are used in order to generate high-quality information to improve in vitro production techniques in small ruminants such as sheep, since it should be considered that only the reproductive capacity of female sheep is evaluated. and at the time of making the selection the oocytes must be taken primary care since this will affect the whole process.

According to studies by Zhu et al. [10] showed that advances in in vitro production in sheep by new techniques have improved the entire process to obtain quality oocytes. As there were no studies carried out in sheep, it was decided to compare with the study carried out by Lonergan [11] who, when evaluating P4 in cattle, mentions that this hormone can indirectly affect oocyte quality through its effects on pulsatility of LH and the development of a persistent dominant follicle. When comparing these results with the present investigation there are no significant differences with the corpus luteum that generates progesterone to affect the production of blastocysts.

General efficiency

The differences found were not significant (P>0.05) for the relationships between the number of parthenogenetic blastocysts produced according to the viable oocytes collected, the number of parthenogenetic blastocysts produced with the cleaved oocytes, however, for the relationship parthenogenetic blastocysts produced by There were significant differences (P≤0.05), with the treatment with the corpus luteum being the one that obtained the highest values, surpassing the treatments without corpus luteum and pregnant women in 73.48% and 70.98% respectively (Table 2), which shows that the stage physiological condition in which the ovary is at the moment of being aspirated, if it influences the number of oocytes aspirated, the viability and the number of parthenogenetic blastocysts obtained.

Table 2:Mean values for the general efficiency of the procedure in relation to blastocysts: viable oocytes; blastocysts: cleavage oocytes and blastocysts: ovary in oocytes from sheep ovaries according to the physiological condition of ovaries with corpus luteum, without corpus luteum and pregnant.

^abAverage values in columns with different letters, differ statistically from each other (P≤0.05).

Therefore, based on the results obtained, the most advisable thing would be to aspirate only ovaries that present corpus luteum, however, the difficulty would lie in being able to exclusively acquire this condition, since in slaughter plants the sheep enter indistinctly at any stage of the estrous cycle, however, another alternative would be to perform heat synchronization processes prior to sacrifice or laparoscopically guided follicular aspiration, thus achieving that a group of sheep can have the corpus luteum as the dominant structure in their ovaries at the time of aspiration.

The quality of the oocyte is one of the factors that most affect the performance and production of blastocysts [12]. The quality of the oocyte is influenced by many factors (race, age, prolificacy of the females), however, one of the main ones is the correct classification of the oocytes, since by using only those of class A and B, and discarding all those of class C and D, a better development and competence of the oocytes in the process of maturation and in vitro fertilization can be expected, which would mean a higher percentage of blastocysts.

The ovaries with the presence of the corpus luteum presented higher production of parthenogenetic blastocysts, that is, that the physiological stage in which the ovary is at the moment of being aspirated does influence the amount of oocytes aspirated, the viability and the number of parthenogenetic blastocysts obtained.

The authors thank the Meyer Hall Reproduction Laboratory in the Department of Animal Science, at the University of California, Davis in the United States and the Animal Reproduction Biotechnology Laboratory at the University of Zamorano, Honduras for their support both in the logistics part as funding for this research.

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