Introduction

IVP(Invitro Production) is performed to transfer, freeze and other technologies such as cloning, transferring gene from an animal to other animals and also maintaining a genetically superior animals.Invitro production in cattle has four steps which included: taking oocytes from follicular, oocytes maturation in the laboratory or IVM (Invitro  Maturation), IVF (Invitro Fertilization)  and IVC (Invitro Culture).Obtaining the oocyte and embryo productionis used in non-pregnant cows and heifers, pregnant cows to day 110 of pregnancy, cows that do not respond to injections of FSH for superovulation and also cows with reproductive problems such as blockage of the fallopian tubes in the lab¹.

IVF related problems can be cited polyspermia at the time of IVF, large loss of embryos 35 days after embryo transfer that most deaths related to lack of development in allantois².The most serious problemin IVF will be large size and abnormality of fetus.In this regard, there are some reports indicate respiratory problems, sudden death near the birth of calves and disinterested with regard to breastfeeding³.

The total number of embryos produced by IVP in cattle to 2011 was equal to 453 471 pcs which included embryos from oocytes obtained by ovum pick-up (OPU) and slaughterhouses⁴.The number of oocytes taken from cows ovaries can be affected by genetic, diet and weather conditions^5,6.In a new study has emphasized the role of nutrition in the number of oocytes, embryo quality of their^7,8.

There are several ways to collect follicular oocytes that are laparoscopy, surgical removal of ovarian, ovaries collected from slaughterhouse and Ovum Pickup(OPU).For the first time in 1987inDenmark was raised guiding ultrasonic and ovarian follicular aspiration⁹.In 1988, in Netherlands was the first real OPU¹⁰.

Generally maintenance of immature oocytes before the onset of puberty helps to do next manipulation¹¹.Also culture in the maturation inhibitors such as roscovitine and butyrolactone Ican be effective in keeping bovine oocyte in germinal vesicle stage^12,13, but it may be reduced blastocyst development¹⁴. These medias can be toxic if used long. So it is necessary to used in shorter time or environments that Have the least impact on cytotoxicity and best effect in oocyte maintenance¹¹.

According to the above-mentioned, there is a need for using the environment tomaintenance and preservation of oocytes in the process of obtaining oocytes and sending it to the laboratory for IVF and embryo transfer, in the short and long distances.Despite the losses and damage caused by the media holding it is advisable to use them for storing oocytes. So it should be used media  holding where have minimal damage and negative impact on the process of sampling and sending to the laboratory for completion.On the other hand it is possible to use accessories like incubators in the process of doing cell work. While the operation is performed on the farm,it is not possible completely.In this research we decided touse Embryo Holding (Syngro^@)with and without the use of the incubator for oocyte transfer to the laboratory and examining the positive and negative effects on oocytes.

Materials and Methods

Oocyte Recovery

Cow ovaries were collected from slaughterhouse animal and were transported to the laboratory less than 1 hour. Oocyte – Cumuluscomplex  were aspirated of follicles 2-6 mm size byneedle 18G and syringe. PBS supplemented with penicillin and 0.2% 0 BSA were used for storage the recovery procedure¹¹.

Design of Experiments

Oocyte – Cumuluscomplex were classified into 7 randomized groups.  6 groups (EHT) of them were replaced on the Embryo Holding (Syngro^@_ USA) and kept in darkness in a styrofoam container with thermo packs at 4°C, 22°C, 38.5°C for 12, 24 hours ( EHT-12h, EHT-24h ).  1 group was replaced on the standard medium IVM.

IVM ( Invitro Maturation )

Maturation medium consisted of TCM-199 with Earle ^,s salt supplemented with 5% estrous cow serum, 3.5 µg/ml FSH, 3.5 µg/ml Hcg (Sigma-Aldrich) and 1 µg/ml estradiol 17β. Maturation medium was carried out under mineral oil at 38.5°C in 5% CO₂ for 22 hour¹¹.Treatment groups were transferred to the IVM media (mentioned above) after the desired time the Holding.

Determination of Oocyte Maturation

Oocytes of each group were stripped of cumulus cells in working medium containing hyaluronidase (0.01% w/v) under mineral oil and were evaluated for the presence of a polar body under the stereomicroscope.Then oocyte will be fixed in 4% formaldehyde and stain with Hoechst 33342 (Sigma-Aldrich) for their nuclear maturation status¹¹.

Recording And Analysis of Data

The data was transfered to spss:19. The normality of distribution of maturation was checked using tests of normality. One-way ANOVA was used to compare dependent variable (maturation) in different groups with P ≤ 0.05 considered to be significant.

Results and Discution

According to the results in table.1, the highest percentage of oocytes maturation was 92/2 % in the control group (C₁) while it had significantly different from all groups experiment (P≤0/05).

EHT-12h numerically had the highest percentage of maturationbetween treatment groups in 22°C and 38.5 °C (80%,79.5%) and they didn’t have significant difference (P>0/05). Also the lowest percentage of oocytes maturation was in 4°C (71/6 %) which had no statistically significant difference from EHT-12h-22°C and EHT-12h-38/5°C (P>0/05).

In EHT-24h, The highest and lowest percentage of oocytes maturation was in 38.5 °C and 4°C (54.3% vs. 38%) and they had statistically significant difference (P≤0/05). Also, there was no statistically significant difference between EH-24h-4°CwithEHT-24h-22°C and EHT-24h-22°C with EHT-24h-38/5°C (P>0/05).

According to table.1 , the highest percentage of immature oocytes and degenerated was observed in EHT-24h-4°C (62%) which had difference statistically significant with C₁ group(7.8 %, P≤0/05).Statistical analysis showed that oocytes maturation in EHT-12h was more than EHT-24h and there was significant differences ( P≤0/0001 ).

Table 1: Determination of oocyte maturation after EH treatment (n total = 721 oocytes)

The aim of this study was maintenance of oocytes obtained from slaughterhousein the Embryo Holding (Syngro^@ ). As it is clear because of the distance andtime from the slaughterhouse to the laboratory , oocytes obtained are changed that can reduce the maturation and ultimately reduce the rate of blastocyst. As well as the purchase and transportation incubator that can keep the oocytes is expensive and hard. Therefore, there is the need for beneficial holder media without the need for incubator.

In the past, various studies have been done to maintain the immature oocytes. In a study byusing  Butyrolactone I (B) as meiosis inhibition could block meiosis for 24 hoursand then compered the oocytes maturation with the control group after IVM.The results showed that 95% of oocytes that were blocked in the first stage, progressed to metaphase II and had no difference from the control group. It also stated that this inhibitor can be used for storing oocytes before IVM. The results are listed in studywas not consistent with our results in 12 hours holding at 4°C, 22°C and 38.5°C.But in a last study the rate of blastocyst in treatment group (B) was less than the control group¹³.

Ponderato and et al.¹⁶, showed that the use of different doses of Butyrolactone I (B) and Roscovitine (R) have different impact on block meiosis and progressed to metaphase II. Of course, they thought that high doses can have toxic effects on the oocytes.

Adona and et al.¹³, used B and R to suppress and control the accelerationmeiosis.Given that these substances can increase the speed of nuclear maturation after the transition to media maturation, so they were added to the TCM-199with low doses and used for the maintenance of immature oocytes. Before starting IVM in zero hour, in the group which only B was added GV stage was to maintain a higher percentage than the group BR (100% vs. 89%, P≤0/05). They concluded that group B had less effective in blocking meiosis. Also treatment groups with B and BR showed more speed to the resumption of meiosis in maturation than the control group.

Our study showed that maintenance of immature oocytes in the EH, for 12 hours at temperatures 4°C, 22°C, 38.5°C made maturation to 71.6%, 80% , 79.5% after IVM which is significantly different from the control group (92.2%, P≤0/05). Amongtemperatures tested, 22°C and 38.5°C have similar results during the 12 hours maintenance. But 22°C (room temperature, no incubator) is preferable to 38.5°C (with incubator). In three temperatures, 4°C has the lowest rate of oocyte maturation which could be due to the bovine oocyte sensitivity to temperature reduction. The results also showed that maintenance of immature oocytes in EH for 24 hours at 38.5°C showed 54.3%  maturation that compared with EHT-24h-4°C andEHT-24h-22°C was higher (38%, 38.5%) and compared with control group was so lower (92/2%, P≤0/05).

In 2008, Alm et al.¹¹, expressed a new idea while without the use of inhibitors meiosis keptimmature oocytes at room temperature for 18 hours then entered maturation media. Holding used in their study was 40% TCM-199 with Hanks’salts ,40% TCM-199 with Earle’ s salts 25 mM Hepes and 20% FBS (fetal bovine serum). After the transition to maturation media cheched chromatin status at different times. At zero hour, there was no statistically significant difference between treatment groups and control in maintaining GV stage. (79.3% vs. 87.5%, P>0.05). Alm’s results had significant differences from this study. Nuclear maturation in this study in EHT-12h,at 4°C, 22°C,38.5°C (71.6%,80%,79.5%) was higher Alm’s results at 14 hours holding in room temprature. Therefore, holding media we used has better maturation after IVM while in Alm’s results maintenance of immature oocytes up to 24 hours had better maturation than our study for 24 hours at 22°C (69% vs. 38.5%). The percentage of nuclear maturation in EHT-24h-38.5°C was less than Alm’s study(54.3% vs. 69%).

As mentioned, few studies have been carried out for immature oocytes maintenance before the IVM. In some cases, oocyte freezing was used as a quick method for long term storage of oocytes, but greatly reduced the development of cow’s oocytes.^16,17Oocyte aging process reduced the activity of MPF (maturation promoting factor), spontaneous activation causingcortical granule exocytosis , granules movement disorder, changes in mitochondrial function and ATP, zona hardening and apoptosis. Finally decline its growth and developmentthe effect.^18,19

Studies in mouses showed that temperature affected the protection and authority of the metaphaseII, and the right temperature to preserve mature oocytes in mouse is 25-27°C¹¹. In addition there are various differences between species in sensitivity to store oocytes between cattle and mouse. Cytoplasmic features and microtubules lipid affect between two species²¹.Microtubules have a key role in the mobility and distribution of mitochondria in the cytoplasm. They are the main structure of meiotic spindle in oocytes.²² Studies showed that bovine oocyte serves more sensitive toreduce the temperature by virtue of having large amounts of fat compared to mouse. In the present study we were able to record 71.6% maturation in EHT-12h-4°C. In this study was observedless impact of cold on oocytes and can be considered as one of the advantages of embryo holding.

The groups EHT-12h-22°C (80%) and EHT-12h-38.5°C (79.5%) had more  maturation than 4°C (71.6%). We concluded that using EH for immature oocytes maintenance at 22°C and 38.5°C during 12 hours coused acceptable maturation in oocytes.

The researchers argued high phosphate levels involved during the cells of PH and calcium homeostasis. Therefore, the use of TCM-199 is superior to PBS due to lower phosphate levels. However, inhibitors meiosis like B and R coused maintenance of GV stage but high doses can have toxic adverse effects on the oocytes and ultimately the rate of blastocyst.¹¹

Conclusion

EH media in our testing showed that phosphate and harmful substances can be helpful for maintenance of immature oocytes until 12 hours at room temperature,but most importantly,there is no need for incubation in experiment.

It is good tobe considered IVF treatment and the rate of blastocyst at the time listed in other studies while we did not discuss it.This results in the future could be a great help in the process of IVM, IVF and embryo production.

Acknowledgements

The authors thank to Dr.Pouya Dini, FKA husbandry FKA agri-animal production co., Dr. Nima Sadeghi Borujeni, Dr. Mahdi Safahani , Mr. Jalilnejad for their helps during this study.

Refrences

1. Robert, S.Youngquist, Walter, R., Threlfall. : Current Therapy In Large Animal Theriogenology, Second Edition: 497.
2. Thompson, J.G. & Peterson, A.J. Bovine embryo culture in vitro: new developments and post-transfer consequences. Human Reproduction,. 2000; 15, (5): 5967.
   CrossRef
3. Byrne, A.T., Southgate, J., Brison, D.R., Leese, H.J. Regulation of apoptosis in the bovine blastocyst by insulin and the insulin-like growth factor (IGF) superfamily.Molecular Reoroduction and Development, 2002; 62, 489-495.
   CrossRef
4. Stroud B. IETS Statistics and Data Retrieval Committee Report. The year 2011 worldwide statistics of embryo transfer in domestic farm animals. 2011; IETS Newsletter.
5. Meiyu Qi, Yuchang Yao, et al. Transvaginal Ultrasound-guided Ovum Pick-up (OPU) in Cattle. Biomim Biomater Tissue Eng, 2013; 18: 118.
6. Wolfenson, D., Roth, Z., Meidan, R. Impaired reproduction in heat-stressed cattle: basic and applied aspects. Anim Reprod Sci., 2000; 60-61: 535-547.
   CrossRef
7. Leroy, J.L., Rizos, D., Sturmey, R., Bossaert, P., Gutierrez-Adan, A., VanHoeck, V., et al. Intra follicular conditions as a major link between Maternal metabolism and oocyte quality : afocus on dairy cow fertility. Reprod Fertil Dev,. 2011; 24: 1–12.
   CrossRef
8. Leroy, J., VanSoom, A., Opsomer, G., Goovaerts, I., Bols, P. Reduced fertility in high-yielding dairy cows: are the oocyte and embryoin danger ?PartII. Mechanisms linking nutrition and reduced oocyte and embryo quality in high-yielding dairy cows. Reprod Domest Anim, 2008; 43: 623–32.
   CrossRef
9. Callesen, H., Greve, T., Christensen, F., Ultrasonically guided aspiration of bovine follicular oocytes. Theriogenology, 1987; 27: 217.
   CrossRef
10. Pieterse, M.C., Vos, PLAM., Kruip, TAM., Wurth, Y.A., Van Beneden, T.H., et al. Transvaginal ultrasound guided follicular aspiration of bovine oocytes. Theriogenology, 1991; 35: 857–862.
    CrossRef
11. Alm, H., Choi, Y.H., et al. Holding bovine oocytes in the absence of maturation inhibitors:Kinetics of invitro maturation and effect on blastocyst Development after invitro fertilization. Theriogenology, 2008; 70: 1024-1029.
    CrossRef
12. Adona, P.R., Pires, P.R.L., Quetglas, M.D., Schwarz, K.R.L., Leal, C.L.V., Nuclear maturation kinetics and invitro embryo development of Cattle oocytes premature with butyrolactone I combined or not Combined with roscovitine. Anim Reprod Sci, 2008; 104: 389–9.
    CrossRef
13. Lonergan, P., Dinnyes, A., Fair, T., Yang, X., Boland, M., Bovine Oocyte and embryo development following meiotic inhibition With butyrolactone I. Mol Reprod Dev, 2000; 57: 204–9.
    CrossRef
14. Ponderato, N., LagutinaI., Crotti, G., Turini, P., Galli, C., Lazzari, G,. Bovine oocytes treated prior to invitro maturation with a Combination of butyrolactone I and roscovitine at low doses Maintain a normal developmental capacity. Mol Reprod Dev, 2001; 60: 579–85.
    CrossRef
15. Ponderato, N., LagutinaI, Crotti, G., Turini, P., Galli, C., Lazzari, G,. Bovine oocytes treated prior to invitro maturation with a Combination of butyrolactone I and roscovitine at low doses Maintain a normal developmental capacity. Mol Reprod Dev, 2001; 60: 579–85.
    CrossRef
16. Martino, A., Songsasen, N., Leibo, S,. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod, 1996; 54(5) :1059-69.
    CrossRef
17. Otoi, T., Yamamoto, K., Koyama, N., Tachikawa, S., Suzuki, T., Cryopreservation of mature bovine oocytes by vitrification in straws. Cryobiology, 1998; 37(1): 77-85.
    CrossRef
18. Yanagimachi, R., Chang, M., Fertilizable life of golden hamster ova and their morphological changes at the time of losing fertilizability. J Exp Zool, 1961; 148(3): 185-203.
    CrossRef
19. Wilcox, A.J., Weinberg, C.R., Baird, D,D., Post-ovulatory ageing of the human oocyte and embryo failure. Hum Reprod, 1998; 13(2): 394-7.
    CrossRef
20. Wang, T.Y., Li, Q., Li, Q., Li, H., Zhu, J., Cui, W., et al. Non-frozen preservation protocols for mature mouse oocytes dramatically extend their developmental competence by reducing oxidative stress. Mol Hum Reprod , 2014; 20(4): 318-29.
    CrossRef
21. Suttirojpattana, T., Somfai, T., Matoba, S., Nagain, T., Parnpai, R., Geshi, M. The effect of temperature during liquid storage of in vitro matured bovine oocytes on subsequent embryo development. Theriogenology, 09.033.
22. Sun, Q., Wu, G., Lai, L., Park, K., Cabot, R., Cheong, H., et al. Translocation of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro. Reproduction, 2001; 122(1): 155-63.
    CrossRef