Research Influence Heat Shock on the Quality of Post Goat Oocytes Warming Through Evaluation Maturation Rate and Inspection Protein Heat Shock (HSP-70), Adenosine Triphosphate (ATP), Glutathione (GSH)

doi:10.21203/rs.3.rs-5240067/v1

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Research Influence Heat Shock on the Quality of Post Goat Oocytes Warming Through Evaluation Maturation Rate and Inspection Protein Heat Shock (HSP-70), Adenosine Triphosphate (ATP), Glutathione (GSH)

https://doi.org/10.21203/rs.3.rs-5240067/v1

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Local goat cultivation in Indonesia faces challenges due to imports of domestic goats, which threaten the genetic variation of local goats. To overcome this, veterinarians are directed to play a role in biotechnology, especially through the In Vitro Fertilization (IVF) method. This research is expected to show the identification of the three biomarkers, namely ATP, GSH, and HSP-70 from post maturation goat oocytes exposed to two different types of cryoprotectant, namely commercial cryoprotectant (Cryotech) and Ethylene glycol 30%+1M sucrose as well as comparing the quality of the goat oocytes after ELISA examination. This study used a completely randomized design with three groups of mature goat oocytes, consisting of one control group and two vitrification treatment groups with different cryoprotectants. Goat ovaries were collected from the Pegirian Surabaya slaughterhouse, then the oocytes were selected and matured in a CO2 incubator. The results showed that there was no significant difference in the maturation rate between the three groups of goat oocytes (control, Cryotech, and EG30%+1M Sucrose). However, there were significant differences in HSP70, GSH, and ATP levels. The EG30%+1M Sucrose group (Treatment 2) showed the lowest levels of HSP70 and GSH, but the highest levels of ATP compared to the other groups. This investigation demonstrates no significant difference in caprine oocyte maturation rates among the control and two cryoprotectant treatment groups. However, the EG30%+1M Sucrose treatment significantly altered HSP70, GSH, and ATP levels. Both cryoprotectants exhibited comparable efficacy in preserving oocyte quality during vitrification.

ATP

Goat Oocytes

GSH

HSP-70

Zero Hunger

It is reported that local goat cultivation in Indonesia is starting to become rare due to the influence of information and the positive influence of breeders in importing domestic goats from abroad. As a result of this influence, new problems emerged that made veterinarians aware that local goat genetic variations were given little attention and almost no pure genetics were obtained. To avoid the extinction of the pure genetics of local goats, veterinarians are directed to play a high role in the world of biotechnology so that the genetic preservation of local goats in Indonesia can be quickly secured [1].

In Vitro Maturation is a crucial part of the method In Vitro Fertilization in the world of biotechnology because it aims to secure and change the quality of oocytes that are still primary follicles into mature oocytes, where this process is carried out in a special and sterile laboratory. This crucial process of oocyte maturation involves selecting an appropriate and safe IVM medium, regulating humidity and temperature levels, regulating antioxidants and growth factor for oocyte growth and development [2].

Generally, after the fertilization process between sperm and oocytes continues with the embryo transfer process to the mother. However, this causes problems if the recipient does not get the mother or another problem is that the goat has a reproductive disorder other than the ovaries, the follicles are still developing well and want to be protected. So, a new process emerged that was developed specifically to safeguard oocytes, not only in goats, for all types of animal oocytes which are similar to the process such as artificial insemination for sperm and this process is part of cryopreservation, referred to as vitrification (super fast freezing of animal oocytes) [3].

Oocyte vitrification is applied according to the request of researchers, from several references, oocytes can be frozen in an immature or mature condition. However, for this study, oocytes were frozen after going through the maturation process in vitro. Vitrification includes several components, namely liquid nitrogen and cryoprotectant. The type of cryoprotectant used contains high concentrations and can damage the oocyte structure. So far, the cryoprotectants used to freeze oocytes are dimethyl sulfoxide, ethylene glycol, propanediol, and glycerin. Their function is to protect the oocyte so that it is not 'shocked' when exposed to temperature liquid nitrogen − 196°C [4]. From maturation to freezing, the oocyte continues to activate its metabolism and the resulting products are used and exist as waste products (free radicals) called reactive oxygen species (ROS). To prevent waste products from damaging other metabolism, oocytes secrete endogenous antioxidants such as heat shock protein (HSP70) [5], glutathione (GSH) to protect oocyte mitochondria. Then adenosine trihydrate (ATP) is the energy needed by mitochondria to produce energy, new molecules and DNA for oocyte maturation [6]. If ROS damage the metabolism of each part of the oocyte to the point of a lack of ATP, the growth and maturation of the oocyte will be disrupted, causing metaphase II not to occur in a balanced manner [7].

Experimental Design

This study used a completely randomized design system using three groups of oocytes, each of which had been matured. One group was a control group that was not vitrified (no treatment) and two groups had vitrification treatment by being exposed to two different types of cryoprotectants [7].

The research was carried out from June 2023 to March 2024 at three research locations, namely the Pegirian Slaughterhouse, Surabaya, Indonesia; In Vitro Laboratory, Faculty of Veterinary Medicine, Airlangga University, Surabaya, Indonesia; and Genetics Laboratory, Faculty of Science and Technology, Airlangga University, Surabaya, Indonesia.

Ovarian Collection

Goat ovaries were obtained from the Pegirian Slaughterhouse, Surabaya, Indonesia which were stored in a thermos containing physiological NaCl solution (0.9%) at a temperature of 37°C and antibiotics then delivered to the laboratory. In Vitro, Faculty of Veterinary Medicine, Airlangga University, Surabaya, Indonesia. The ovaries were rinsed using sterile phosphate buffer solution (PBS), then the follicles were aspirated using a 10 mL syringe containing 1 mL sterile PBS and an 18G needle. The ovarian follicles collected were sized 2–6 mm [6].

We select complex cumulus oocytes during the selection process using a microscope inverted ECLIPSE Ts2 (Nikon, Japan). The oocytes selected are grade A (complete COC) and grade B (complete COC with 2 or 3 layers of cumulus) [5].

Oocyte Maturation

Selected complex cumulus oocytes are placed in maturation media, Universal IVF Medium and soaked using a mineral oil solution (FertiCult™). The maturation medium containing oocytes was placed in a 5% CO2 incubator, 98% humidity, and a temperature of 38.5°C for 22 hours. After the maturation process, oocytes were divided into three groups, namely a control group (not given vitrification treatment) and two vitrification treatment groups using different types of cryoprotectant. Oocyte maturity evaluation was conducted post-incubation for 22 hours, utilizing an Olympus x41 inverted microscope. A denudation procedure was implemented to eliminate cumulus cells and facilitate visualization of the first polar body. Enzymatic denudation was executed using hyaluronidase (HYASE-10x™, Vitrolife®) for a duration of 30 seconds, followed by transference to fresh culture medium Quantification of oocyte maturation levels was based on the frequency of first polar body extrusion, indicative of meiosis I completion and attainment of metaphase II stage [8].

Vitrification Treatment Group 1

Post-maturation goat oocytes are exposed to cryoprotectant from Cryotech®Japan. The exposure procedure is the first exposure equilibration solution with oocytes for 12–15 minutes, then second exposure vitrification solution for 1 minute [8]. Oocytes are placed in hemistraw which is secured with straw 0.5 mm then soaked in liquid nitrogen container for seven days [9].

Warming Treatment Group 1

Frozen samples were removed from the liquid nitrogen container after preparing the media warming Cryotech®Japan by following the procedure stated by the product, namely first exposure by thawing solution for 1 minute, followed by a second exposure with dilution solution with oocytes for 3 minutes, then a third exposure with warming solution for 5 minutes, and final exposure with warming solution 2 for 1 minute. Then oocytes post warming put into an effendorf tube containing 4x sterile lysis buffer [8].

Vitrification Treatment Group 2

Oocytes were exposed to the cryoprotectant 30% ethylene glycol and 1 M sucrose following the procedure of Patent IDP00008642, namely the first exposure to 30% ethylene glycol for 15–18 minutes and the second exposure to 1 M sucrose for 30 seconds. Oocytes are placed in hemistraw packed by straw 0.5 mm, then soaked in a liquid nitrogen container for 7 days [9].

Warming Treatment Group 2

Oocytes were taken back from the container using the procedure from Patent IDP00008642 by sucrose concentrations of 0.25 M, 0.5 M, and 1 M where the first exposure was to a concentration of 0.25 M for 1 minute, then the second exposure to 0.5 M for 2 minutes, and the third exposure to 1 M for 3 minutes. The oocytes were secured into an Effendorf tube containing 4x sterile lysis buffer [10]. ELISA examination of Heat Shock Protein (HSP-70), Adenosine Triphosphate (ATP), Glutathione (GSH).

Post-warming goat oocytes were examined using the ELISA Kit Rat HSP70/HSPAS 9 (Heat Shock Protein 70) (Catalogue: E-EL-R0479 Elabscience), Rat Adenosine Triphosphate (ATP) ELISA Kit (Cat. No. E0665Mo, BT Lab), GSH ELISA Kit (Cat. No:E- EL-0026, BT Lab). The ATP and GSH examination procedure uses an ELISA Kit from the same brand, while the HSP70 examination uses a different brand, starting with adding 50 µl of standard solutions of various concentrations or samples to the ELISA well plate, then adding 50 µl of Biotinylated antibody and incubating for 60 minutes, 37ºC. Next, the well plate was washed 4–5 times with 300 µl washing buffer, then 50 µl of Streptavidin-HRP was added to the standard and sample wells and incubation again, followed by washing the well plate 5 times with 300 µl washing buffer, continued with the addition of 50 µl Substrate A and 50 µl Substrate B in the dark. Then, wait about 10 minutes until it shows a blue color (as a positive response), followed by adding 50 µl of stop solution to show a yellow color. Finally, the OD (optical density) value is read using an ELISA reader at a wavelength of 450 nm and creating a standard curve between the OD value and the standard solution levels to obtain a regression equation to calculate the levels of the sample [11].

Statistical Analysis

All data of oocyte maturation rate with biomarker (ATP, GSH, HSP-70) levels from ELISA detection were subjected to non-parametric variables followed by Mann-Whitney and Kruskal-Wallis test (IBM SPSS Statistics Version 2023), also differences at P < 0.05 were considered statistically significant.

We show the results of the data that were successfully collected in Table 1 maturation rate, HSP70, GSH, and ATP levels of each group of oocytes along with Fig. 1 described the photoshoot of matured goat oocytes.

Table 1

Goat Cumulus Complex Oocytes based on the results of ELISA examination of HSP70, ATP, and GSH.
Group	Maturation Rate	HSP70 Levels	GSH Levels	ATP Levels
Control Group (CG)	84.3%^a	0.83 ± 0.58^a	25.95 ± 1.95^a	0.83 ± 0.58^a
Cryotech (Treatment 1)	79.8%^a	1.56 ± 1.96^a	25.65 ± 1.63^a	1.56 ± 1.96^a
EG30%+1M Sucrose (Treatment 2)	77.2%^a	0.07 ± 0.01^b	52.13 ± 7.70^b	0.07 ± 0.01^b
*Different letters within each column indicate a significant difference (P < 0.05)

Table 1 shows the effect of treatment on ripening rate and several cellular parameters. There were three treatment groups: Control (CG), Cryotech (Treatment 1), and EG30%+1M Sucrose (Treatment 2). Maturation rate did not show significant differences between groups, with values ranging from 77.2–84.3%. HSP70 levels showed significant differences between groups. The control group had the highest HSP70 level (0.83), followed by Treatment 1 (1.56), while Treatment 2 had a very low HSP70 level (0.07). GSH levels also showed significant differences between groups. Treatment 2 had the lowest GSH level (0.07), while the control group and Treatment 1 had similar GSH levels (approximately 0.83 and 1.56). There was a significant difference in ATP levels between the control group and Treatment 2, with Treatment 2 having a higher ATP level (52.13) than the control group (25.95). There were no significant differences in ATP levels between the control group and Treatment 1. Overall, the results showed that Treatment 2 had a significant impact on HSP70, GSH, and ATP levels, while not significantly affecting the ripening rate.

Based on Table 1, the maturation rate section shows that there is no difference between the three groups, giving the possibility that goat oocytes exposed to the two cryoprotectants produce the same oocyte quality effectiveness. The success of high quality goat oocytes is influenced by the success of the in vitro maturation process of goat oocytes [11]. Oocyte maturation encompasses nuclear maturation and synchronized cytoplasmic modifications essential for fertilization and early embryonic morphogenesis. Nevertheless, a primary challenge in oocyte in vitro maturation (IVM) is the asynchrony between nuclear maturation and cytoplasmic alterations. Invariably, nuclear maturation following an oocyte IVM protocol is readily discernible, as it involves germinal vesicle breakdown (GVBD) and the extrusion of the initial polar body (PB). However, it is well-established that during cytoplasmic oocyte maturation, Ca2 + oscillations are crucial for modulating a wide spectrum of physiological processes. For instance, these oscillations can inhibit GVBD in mammalian oocytes, at least until the first metaphase, thereby preventing spontaneous intracellular meiosis resumption in vitro [12].

The in vitro oocyte maturation process is influenced by various types of metabolism which involve increased ATP production to produce further products to be used for proliferation and differentiation of oocytes as well as cumulus cells until the discovery of GSH and HSP70 to protect oocytes from shock caused by metabolic waste products, ROS, from the metabolic system is working and pH as well as temperature. Heat-shock proteins 70 mediate regulating various physiological processes, including folliculogenesis, oogenesis, or embryo development. HSPs have been shown to promote cell survival by inhibiting cell apoptosis. However, exposure to strong heat stress increases cell susceptibility to apoptosis compared to mild exposure [13, 14]. While, (GSH)-cycling system play key roles in the regulation of cell fates, including proliferation, differentiation and apoptosis, via various signaling molecules and pathways. However, the excessive accumulation of ROS results in intracytoplasmic oxidative stress, affecting molecules, cell structures and protein functions [15].

Vitrification is a super-fast freezing method for oocytes by soaking them with a high concentration of cryoprotectant to protect the structure of oocyte components from the formation of ice crystals, so that damage to the oocyte membrane does not occur [16]. We used these two cryoprotectants, namely Cryotech cryoprotectant and homemade modified cryoprotectant to compare the quality results of goat oocytes after vitrification because we hope that this modified cryoprotectant can be used on a mass scale specifically for vitrifying goat oocytes. Cryotech's cryoprotectant content contains ethylene glycol, dimethyl sulfoxide, HEPES media, and trehalose while the modified cryoprotectant contains 30% ethylene glycol and 1 M sucrose. Generally, ethylene glycol as a cryoprotectant has high permeability properties with a molecular weight of 62.07 g/mol so it easily diffuses across cell membranes, safer than using DMSO, PROH, or glycerol [17, 18]. Then, we used 30% concentration of ethylene glycol with 1 M sucrose. It is known that these two types of molecules balance each other to protect oocytes and increase the percentage of viability of cow and goat oocytes to above 50% [19].

The simple basis for vitrification is that most oocytes can experience damage resulting from changes in temperature (normal temperature to extreme freezing temperature or extreme temperature returned to normal temperature), viscosity, and fluid osmotic changes, such as in the freezing-warming process, this is a change in oocyte fluid. and cryoprotectant liquid [20]. Research has demonstrated that certain cryoprotective agents, including dimethyl sulfoxide (DMSO) and ethylene glycol (EG), induce a temporary elevation in cytoplasmic Ca2 + concentrations across diverse cellular lineages. Additionally, the exocytosis of cortical granules with the oocyte's plasma membrane is similarly Ca2+-reliant. This observation suggests that these cryoprotectants stimulate cortical granule exocytosis by elevating intracellular Ca2 + levels. Contemporary investigations have elucidated that DMSO and EG provoke substantial transient augmentations in cytoplasmic Ca2 + within murine oocytes, precipitating zona pellucida hardening and markedly diminishing fertilization efficacy. Limited studies have juxtaposed the vitrification procedure pre- and post-in vitro maturation (IVM). It has been established that vitrified immature oocytes can undergo post-thawing IVM and fertilization, generating embryos capable of withstanding vitrification and warming without inducing chromosomal aberrations. The subsequent inquiry pertains to whether IVM potential is compromised due to the cryopreservation process in oocytes vitrified at the germinal vesicle (GV) stage. DMSO has been demonstrated to elicit a transient cytoplasmic Ca2 + elevation across various cellular lineages, while elevated concentrations of EG (10–40%) similarly increase intracellular Ca2 + in murine oocytes. Consequently, a potential complication in oocyte cryopreservation is the induction of a primary activation event. DMSO appeared to induce a more pronounced and sustained Ca2 + elevation compared to EG, potentially attributable to the enhanced permeability of oocytes to DMSO. The permeability of oocytes to cryoprotectants and their capacity to substitute intracellular water is another well-documented phenomenon. The lipophilic properties of DMSO and EG are expected to exert non-specific effects on the plasma membrane and other intracellular membranes, such as the endoplasmic reticulum (ER), potentially leading to Ca2 + influx and/or Ca2 + release from intracellular reservoirs. However, the osmotic contraction induced by cryoprotectants may also contribute to the elevation in cytoplasmic Ca2 + concentrations [21]. The relationship between ATP, HSP-70, and GSH results from the emergence of free radicals which influence changes in calcium oscillations and cause mitochondrial dysfunction which can disrupt, other active metabolism in the oocyte. Free radicals (ROS) are a waste product of oxidative phosphorylation in mitochondrial cells when making ATP [21]. Oxidative stress is induced from the endoplasmic reticulum, then released into the oocyte cytoplasm, then absorbed by the mitochondria, but if excessive amounts of these ions are taken up then the apoptotic pathway is activated [22, 23]. As a result, ROS, oocytes initiate the activation of enzymes and gene factors such as Heat Shock Protein-70 (HSP-70) [24, 25]. As the results of our study found that the P2 group responded to higher HSP-70 production compared to the other two groups having every possibility. The sample responds to stress from osmotic changes between the exchange of cryoprotectant and the oocyte's own fluid during vitrification and warming [26].

Heat Shock Protein 70 (HSP-70) associates with hydrophobic regions of polypeptide chains, functioning as a protective barrier to mitigate undesired aggregation of polypeptide chains, such as those induced by reactive oxygen species (ROS). Depletion of HSP-70 can lead to protein synthesis dysfunction due to the presence of the Hip protein, which interacts with the ATPase domain of HSP-70 to maintain the recycling of polypeptide synthesis emerging from the ribosome. This interaction facilitates the exchange of the nucleotide exchange factor (NEF) from the ADP-bound N-domain to the ATP-bound state. This process is coupled with ATP hydrolysis, which occurs through interaction with HSPs, co-chaperone HSP-40 via its J domain, and subsequently with HSP-60 to orchestrate polypeptide synthesis or protein denaturation. This cascade ensures proper protein folding and perpetuates the ATPase cycle [27].

Specifically, glutathione (GSH) is the main non-protein sulfhydryl compound in mammalian cells, providing protection against oxidative stress-induced damage to spindle morphology and function during bovine oocyte maturation in vitro [26]. Based on the research results, the concentration of GSH in the oocyte cytoplasm is a positive indicator of good cytoplasmic maturation. Several study results state that vitrification can change oxidation-reduction (redox) status, reduce or inhibit glutathione (GSH) levels, and trigger an increase in reactive oxygen species (ROS) levels [27], which has an effect on mitochondrial dysfunction and response. Apoptosis followed by no differentiation and damage to the DNA and oocyte membrane layer [22]. The response of oocytes to osmotic and temperature changes causes an increase in GSH content in mouse oocytes which can affect their tolerance to vitrification [23]. Endogenous GSH inside oocyte happen as essential to protect it against oxidative stress and reactive oxygen stress along with other forms of cellular injury [24].

The equilibrium between ethylene glycol and sucrose in the modified cryoprotectant played a pivotal role in oocyte protection and viability enhancement. This research provides valuable insights into the effects of vitrification on cellular parameters of caprine oocytes, potentially contributing to the development of more effective cryopreservation protocols for application in animal reproduction biotechnology.

The limitations of the study, include: Cryoprotectant Comparability: While the study compares two different cryoprotectants (Cryotech and a modified ethylene glycol + sucrose solution), it restricts the generalizability of the findings to these specific cryoprotectants. Limited Biomarker Focus: The study focuses on three biomarkers (ATP, GSH, and HSP-70). Short-Term Evaluation: The study appears to focus on the immediate effects of vitrification and warming on oocyte quality.

The control group and two cryoprotectant treatment groups (Cryotech and EG30%+1M Sucrose) had similar caprine oocyte maturation rates of 77.2–84.3%. However, EG30%+1M Sucrose substantially affected HSP70, GSH, and ATP levels. Both cryoprotectants maintained caprine oocyte quality throughout vitrification at similar levels, affecting cellular metabolism to protect oocytes from oxidative stress and osmotic fluctuations. The improved cryoprotectant (EG30%+1M Sucrose) showed promise.

ATP

Adenosine Triphosphate

GSH

Glutathione

HSP-70

Heat Shock Protein 70

ELISA

Enzyme-Linked Immunosorbent Assay

CO₂

Carbon Dioxide

IVM

In Vitro Maturation

DNA

Deoxyribonucleic Acid

ROS

Reactive Oxygen Species

COC

Cumulus-Oocyte Complex

Optical Density

GVBD

Germinal Vesicle Breakdown

DMSO

Dimethyl Sulfoxide

PROH

Propylene Glycol

Acknowledgments

The researchers would like to thank the Indonesian Ministry of Research, Technology and Higher Education for funding through the DRPTM fundamental research scheme, and the Institute for Research and Community Service, Airlangga University, Indonesia.

Authos’s Contribution

WW: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing e original draft,Visualization, Supervision, Project administration, Funding acquisition. ND: Methodology, Validation, Investigation, Resources. VFH: Methodology, Validation, Investigation, Resources. SFT, ZS: Writing e original draft, Visualization, Writing e review & editing.

Funding

The basis for this research was funded by DRPTM Kemdikbudristek No.114/E5.PG.02.00.PL/2023 and LLDIKTI RI No.1274/UN3.LLPM/PT.01.03/2023.

Data availability: N/A

Declarations: The authors declare no financial or competing interests.

Ethical approval: Ethical was obtained from the Animal Care and Utilization Committee, Faculty of Veterinary Medicine, Airlangga University. No.1. KEH.051.03.2023.

Consent to Participate: All the authors consent to participate in publication.

Consent for publication: All the authors consent to publish the manuscript.

Competing interests: The authors declare no competing interests.

Cao B, Qin J, Pan B, Qazi IH, Ye J, Fang Y, Zhou G. 2022. Oxidative Stress and Oocyte Cryopreservation: Recent.
Dalton CM, Szabadkai G, Carroll J. Measurement of ATP in single oocytes: impact of maturation and cumulus cells on levels and consumption. J Cell Physiol. 2014;229(3):353–61.
Edashige K. The movement of water and cryoprotectants across the plasma membrane of mammalian oocytes and embryos and its relevance to vitrification. J Reprod Dev. 2016;62(4):317–21.
Kresna A, Widjiati W, Damayanti T. Combination of cryoprotectants ethylene glycol and propanediol on the viability of mouse blastocysts after vitrification. J Physics: Conf Ser. 2019;1146:012028.
Rahman NSA, Khan NAMN, Eshak Z, Sarbandi MS, Kamal AAM, Malek MA, Abdullah F, Abdullah MA, Othman F. 2022. L-Glutathione Exogen Meningkatkan Hasil Vitrifikasi pada Murine Embrio Praimplantasi. Antioksidan (Basel, Switzerland). 11 (11), 2100.
Rienzi L, Gracia C, Maggiulli R, LaBarbera AR, Kaser DJ, Ubaldi FM, Vanderpoel S, Racowsky C. Cryopreservation of oocytes, embryos and blastocysts in ART: systematic review and meta-analysis comparing slow freezing versus vitrification to generate evidence for the development of global guidelines. Hum reproductive Renew. 2017;23(2):139–55.
Al-Anshori AA, Mustofa I, Safitri E, Mulyati S, Rimayanti R, Luqman EM, Widjiati W. Effect of Alpha Lipoic Acid (ALA) Supplementation on In Vitro Media Oocyte Maturation Goats on Tumor Necrosis Factor Alpha (TNF-α) Expression and Malondialdehyde (MDA) Levels. Int Sci J Adv Paym (IJSCIA). 2023;4(1):41–6.
Dwiningsih SR, Darmosoekarto S, Hendarto H, Dachlan EG, Rantam FA, Sunarjo S, Wiyasa IWA, Widjiati W. Effects of bone marrow mesenchymal stem cell transplantation of tumor necrosis factor receptor-alpha 1 expression, granulosa cell apoptosis, and improved folliculogenesis in endometriosis mouse model. world veterinary Med. 2021;14(7):1788–96.
Jiang Y, He Y, Pan X, Wang P, Yuan X, Ma B. Advances in Oocytes In Vivo and In Vitro Maturation in Mammals. Int J Mol Sci. 2023;24(10):9059.
Widyanugraha MA, Widjiati W, Hendarto H. Effect of Endometriosis on Cumulus ATP, Number of Mitochondria and Oocyte Maturity in Cumulus Oocytes Complex in Mice. The influence of endometriosis on cumulus ATP, total mitochondria and oocyte maturation in the oocyte cumulus complex in mice. Brazilian J Gynecol obstetrics: Brazilian Federation magazine Soc Gynecol Obstet. 2023;45(7):e393–400.
Widyastuti R, Setiawan R, Rizky AA, Syamsunarno, Ghozali M, Saili T, Boediono A. Increasing the viability of female oocytes after vitrification heating using combination of different cryoprotectants and carrier systems. J Veterinary Med. 2018;12(3):57–61.
Jingyu Ren Y, Hao Z, Liu S, Li C, Wang B, Wang Y, Liu G, Liu YD. 2021. Effect of exogenous glutathione supplementation on the in vitro developmental competence of ovine oocytes. Theriogenology, Volume 173, 2021, Pages 144–155,ISSN 0093-691X.
Nursadida M, Widjiati W, Mustofa I, Safitri E, Susilowati S, Suprayogi TW. The Effects of Duration of the In Vitro Maturation Process on the Maturation Level and Apoptosis of Kacang Goat. Veterinarska Stanica. 2024;55(1):79–85.
Saumi Z, Hendrawan VF, Widjiati, Luqman EM. Effects Of Dosage In the combination of ethylene glycol and dimethyl sulfoxide as a cryoprotectant Use of vitrification on the viability of mice (Mus Musculus) after blastocysts Warmup. environmentally friendly. Env Cons. 2022;28(3):39–43.
Molina I, Gómez J, Balasch S, Pellicer N, Novella-Maestre E. Osmotic shock generated by vitrification solutions enhances maturation of immature human oocytes in vitro. Volume 14. Reproductive biology and endocrinology: RB&E; 2016. p. 27. 1.
Moawad AR, Tan SL, Taketo T. Beneficial effects of glutathione supplementation during vitrification of mouse oocytes at the germinal vesicle stage of preimplantation development after maturation and fertilization in vitro. Cryobiology. 2017;76:98–103.
Peraçoli JC, Bannwart-Castro CF, Romao M, Weel IC, Ribeiro VR, Borges VTM, Rudge MV, Witkin SS, Peraçoli MT. High Levels of Heat Shock Protein 70 Are Associated With Cytokines Pro-Inflammatory and Can Differentiate Early and Late Preeclampsia. J Reprod immunol. 2013;10:129–34.
Wrzecińska M, Kowalczyk A, Kordan W, Cwynar P, Czerniawska-Piątkowska E. Disorder of Biological Quality and Autophagy Process in Bovine Oocytes Exposed to Heat Stress and the Effectiveness of In Vitro Fertilization. Int J Mol Sci. 2023;24(13):11164.
Zhao. Melatonin inhibits apoptosis and increases the developmental potential of vitrified bovine oocytes. J Pineal Res. 2016;60(2):132–41.
Widjiati W, Faizah Z, Darsini N, Hendrawan VF, Karima HN, Chotimah C, Sumitro SB, Luqman EM. Identification of caspase 3 intensity in post-vitrification pea goat oocytes using confocal laser scanning microscopy. Adv animation Veterinarian Sci. 2020;8(12):1362–6.
Widjiati W, Faizah Z, Darsini N, Hendrawan VF, Karima HN, Chotimah C, Sumitro SB, Yustinasari LR, Kasman AAMN, Ntoruru JM, Luqman EM. Expression and intensity of calcium (Ca2+) in cumulus-oocyte complexes (COCs) of Peanut goats after vitrification. Pol J veterinary Sci. 2022;25(1):19–26.
Taruna D, Purwanto B, Notopuro H, Utomo B, Widjiati, Herawati L, I'tishom R, Aryati. Effects of High Intensity Swimming on Heat Shock Protein 70, Superoxide Dismutase and Malondialdehyde in Male Rats Rattus norvegicus. J Pharmacognosy. 2022;14(3):524–30.
Tharasanit T, Thuwanut P. Cryopreservation of Oocytes in Domestic Animals and Humans: Principles, Techniques, and Recent Results. Anim (Basel). 2021;11(10):2949.
Trapphoff T, Heiligentag M, Simon J, Staubach N, Seidel T, Otte K, Fröhlich T, Arnold GJ, Eichenlaub-Ritter U. Peningkatan toleransi krio dan potential development of mature mouse oocytes in vitro and in vivo by supplementing donor glutathione before vitrification. mol. Hum reproduce. 2016;22:867–81.
Stamperna K, Giannoulis T, Dovolou E, Kalemkeridou M, Nanas I, Dadouli K, Moutou K, Mamuris Z, Amiridis GS. Heat Shock Protein 70 Improving In Vitro Embryo Yield and Quality from Heat-Stressed Bovine Oocytes. Anim is open access J MDPI. 2021;11(6):1794.
Souza-Cácares MB, Fialho ALL, Silva WAL, Cardoso CJT, Pöhland R, Martins MIM, Melo-Sterza FA. Oocyte quality and heat shock proteins in oocytes from cattle breeds which adapt to tropical regions under different conditions environmental thermal stress. Theriogenology. 2019;130:103–10.

No competing interests reported.

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Research Influence Heat Shock on the Quality of Post Goat Oocytes Warming Through Evaluation Maturation Rate and Inspection Protein Heat Shock (HSP-70), Adenosine Triphosphate (ATP), Glutathione (GSH)

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Abstract

Figures

Introduction

Materials and Methods

Experimental Design

Ovarian Collection

Oocyte Maturation

Vitrification Treatment Group 1

Warming Treatment Group 1

Vitrification Treatment Group 2

Warming Treatment Group 2

Statistical Analysis

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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