How Heat Stress Affects the Functionality of the Ovine Cumulus-Oocyte Complex: Implications for In Vitro Embryo Production

How Heat Stress Affects the Functionality of the Ovine Cumulus-Oocyte Complex: Implications for In Vitro Embryo Production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article How Heat Stress Affects the Functionality of the Ovine Cumulus-Oocyte Complex: Implications for In Vitro Embryo Production Alicia Martin-Maestro, Irene Sánchez-Ajofrin, María Iniesta-Cuerda, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5595754/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 May, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Global population growth requires an increase in food production, particularly meat, with an expected rise in sheep farming. However, climate change challenges livestock management, with heat stress negatively impacting reproductive performance. In vitro embryo production (IVP) in sheep farming is promising, though optimizing embryo quality and efficiency remains challenging. Heat stress impairs oocyte developmental competence, affecting IVP outcomes. This study investigates the effect of season given seasonal variations in temperature and temperature humidity index (THI) and in vitro induced heat stress on oocyte quality and embryo production. In the first experiment, ovaries were collected in the four seasons (winter, spring, summer and autumn) with differences in THI and in the second experiment ovaries were exposed to 30°C (control), 38.5°C, 40°C and 41°C. Results indicate that elevated summer temperatures significantly compromise oocyte and cumulus cell viability, DNA integrity, mitochondrial distribution, and blastocyst quality. These detrimental effects persisted into autumn, likely due to a carry-over effect from summer heat stress. Furthermore, in vitro exposure to temperatures at or above 38.5°C led to marked declines in oocyte quality and blastocyst rates. Understanding these effects is essential for developing strategies to mitigate heat stress and enhance reproductive outcomes in sheep. ovine high temperature oocyte quality embryo quality temperature humidity index Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Driven by growth in developing countries, the world population is projected to reach 8.5 and 9.7 billion people by 2030 and 2050, respectively, thereby requiring around a 70% increase in food production globally 1 . Annual meat production, for instance, would have to grow by over 200 million tons 1 . Particularly in Europe, sheep meat is also expected to increase due to the diversification of meat diets and changes in the population structure 2 . To meet these demands and reduce environmental impact, advances in animal agriculture through reproductive biotechnologies will be necessary to provide increasingly efficient and productive livestock and adapt to climate change and global warming 3 . In this context, the application of advanced assisted reproductive technologies, such as in vitro embryo production (IVP), in the farming sheep industry is highly promising and offers several advantages since it allows the production of embryos from oocytes recovered from unstimulated ovaries, non-fertile females, prepubertal, pregnant, senile, and even dead or slaughtered animals 4 . In addition, IVP facilitates the preservation of valuable biospecimens and provides secure transportation of biological material. Nevertheless, despite substantial improvements, the efficiency and quality of IVP-derived embryos are still one of the main challenges of livestock farming 5 , 6 . Globally, new research into how climatic conditions heavily affect reproduction in domesticated farm animals is gaining interest throughout the agricultural sector 7 . From the standpoint of economic efficiency, the reproductive ability of domestic food animals such as sheep is the most critical trait that may be compromised by climate stressors 8 . Previously, fundamental research into environmental stress during summer and its influence on sheep has provided evidence that heat stress can negatively influence oocyte developmental competence and result in embryonic loss 9 . Heat stress may lead to changes in oocyte membrane properties such as decreased phospholipid polyunsaturated fatty acid content 10 , which are essential for gamete fertility 11 . Furthermore, it may alter the transcript levels of genes involved in oogenesis, folliculogenesis, and embryonic development 12 and impair nuclear and cytoplasmic maturation events such as translocation of cortical granules and cytoskeletal rearrangement 13 that can eventually lead to apoptosis 14 . In addition, heat stress may affect the spatial distribution of mitochondria within the oocyte, possibly due to alterations in the cytoskeleton 15 , as well as the proportion of highly polarized mitochondria and the expression of developmentally important genes 16 , 17 . Moreover, in summer, it is not unusual for sheep IVP systems to endure some periodic reductions in embryo yield 18 . In fact, most laboratories cease their activity during the hottest months. Previously, variability in ovine IVP output throughout the year was attributed to an effect of season, related to photoperiod 19 . However, among the several variables that influence seasonal patterns of blastocyst production is also environmental temperature 20 . Consequently, understanding the effects of high temperatures that prevail throughout the warm season on oocyte quality may be a prerequisite for the successful implementation of IVP 21 . Therefore, in the present study, the first objective was conducted to evaluate the effect of the season (periods with differences in ambient temperature and humidity) in which the sheep ovaries are harvested (winter, spring, summer, and autumn) on oocyte quality and in vitro embryo production. Additionally, we have examined the impact of heat stress through elevated ovarian temperature in an in vitro model to understand better the physiological mechanisms of oocyte damage. Such research may ultimately help develop novel strategies to mitigate the impact of summer heat stress on oocyte quality and protect the integrity of the female germline in small ruminants. Materials and methods All chemicals were acquired from Merck Life Sciences (Madrid, Spain) unless stated otherwise. Experimental Design In the Fig. 1 is shown the experimental design. In experiment 1, to study the effect of season on oocyte quality and developmental competence, adult sheep ovaries were collected twice a month from an abattoir located in south-eastern Spain (Murcia; latitude: 37° 59' 13.34" N; longitude: -1° 07' 48.14" W) receiving sheep from surrounding areas during winter (December-February), spring (March-May), summer (June-August), and autumn (September-November) and transported to the laboratory within 3 h at 30°C in physiological saline (8.9 gr/L NaCl) supplemented with penicillin (0.1 g/L). Immediately after arrival, ovaries were processed. To study maturation rates and oocyte quality parameters, 1049 cumulus-oocyte complexes (COCs) were in vitro matured and further examined. The remaining 1642 COCs were subjected to in vitro fertilization (IVF) and in vitro culture (IVC) to evaluate fertilization potential, embryo development, and blastocyst quality. In experiment 2, to investigate the effect of induced heat stress on oocyte quality and developmental competence, an in vitro model was developed. For this purpose, adult sheep slaughterhouse ovaries were collected post-mortem from the same abattoir as experiment 1 receiving sheep from surrounding areas during late autumn and winter. Afterwards, ovaries were transported in physiological saline (8.9 gr/L NaCl) supplemented with penicillin (0.1 g/L) at 30°C, 38.5°C, 40°C and 41°C and stored for 3 h before processing. Then, the quality and developmental potential of 1509 oocytes was evaluated after in vitro maturation (IVM). All experiments of collection and evaluation of COCs, in vitro maturation and fertilization of oocytes, assessment of fertilized oocytes, embryos and cumulus cells were conducted following the procedures previously described by our working group 22 – 24 . Climate Data and Calculation of Temperature Humidity Index for Heat Stress Assessment Daily observed meteorological data for 2019 to 2020 (time of ovarian collection) on each ovary collection season were obtained from the State Meteorological Agency of Spain for the location of Puerto Tocinos (Murcia). Daily values for maximum temperature measurements and relative humidity (RH hereafter) were used to determine mean values for each variable per season. As previously described by Carabaño et al. 25 , the temperature humidity index (THI) was used to assess the potential for heat stress on sheep at our latitude. The THI was formulated specifically to ruminant species 26 and was examined for seasonal patterns of variability. The THI formula used is shown below, with temperatures in degrees Celsius and RH expressed as percentage: THI ruminant = (1.8 T max + 32)–((0.55–0.0055 RH) (1.8 T max –26.8)) The level of heat stress was considered as: normal ≤ 74; moderate 75–78; severe 79–83; very severe (emergency) ≥ 84 27 . Oocyte Collection and In Vitro Maturation Immature COCs were retrieved from the follicles using a scalpel blade in 2 mL of collection medium (TCM199 medium supplemented with 2.38 mg/mL HEPES, 2 µL/mL heparin, and 4 µL/mL gentamicin). Immediately, the COCs with clear or moderately granular ooplasm surrounded by at least three layers of packed cumulus cells were selected and homogeneously distributed in selection media (TCM199 medium supplemented with 2.38 mg/mL HEPES and 4 µL/mL gentamicin). Then, the COCs were washed in TCM199 and 4µL/mL gentamicin. Subsequently, COCs were homogeneously distributed in 4-well plates with 500 µL of maturation medium: TCM199 and 4 µL/mL gentamicin, 100 µM cysteamine, 10 µg/mL follicle-stimulating hormone (FSH), 10 µg/mL luteinizing hormone (LH), and 10% fetal calf serum (FCS). The maturation medium was covered in mineral oil (Nidacon, Gothenburg, Sweden), and COCs were incubated for 24 h at 38.5°C, 5% CO 2, and maximal humidity. In Vitro Fertilization Groups of approximately 40–45 mature oocytes were placed in four-well dishes containing 500 µL of fertilization medium: synthetic oviductal fluid (SOF; 28 ) supplemented with 10% estrous sheep serum (ESS). Oocytes were subjected to IVF using frozen semen of two rams from the germplasm bank of the "Biology of Reproduction Group" of the Universidad de Castilla-La Mancha (UCLM), which is authorized for the collection and storage of sheep semen (ES008007). Thawed spermatozoa were separated using Percoll© density gradient (45%/90%) and capacitated for 15 min at 38.5°C and 5% CO 2 in fertilization medium. Then, spermatozoa (1 x 10 6 /mL) and oocytes were co-incubated at 38.5°C in 5% CO 2 and maximal humidity. In Vitro Embryo Culture After 18 h post-insemination (hpi), putative zygotes were washed by repeated pipetting and transferred to 25 µL drops (about one embryo per µL) of culture medium (SOF supplemented with 3 mg/mL bovine serum albumin), covered with mineral oil and cultured until day 8 post-insemination (dpi) at 38.5°C in a humidified atmosphere and 5% CO 2 , 5% O 2 and 90% N 2 in air. Evaluation of Fertilization and Embryo Production Rates After IVF, oocytes were fixed in 0.5% glutaraldehyde (v/v) for 15 min at room temperature and stored at 4°C until analysis. To examine sperm penetration, cells were stained with Hoechst 33342 (5 µg/mL) for 20 min at room temperature, washed in phosphate-buffered saline (PBS) supplemented with 0.1% PVA (w/v; PBS-PVA), and then analyzed with 20X augmentation by fluorescence microscopy (Eclipse 80i, Nikon Instruments Europe, Amsterdam, Netherlands). Oocytes containing both female and male pronuclei (regardless of the stage of decondensation) relative to the total number of oocytes matured were considered to be fertilized and were classified as normal (2PN), according to the number of swollen sperm heads and pronuclei in the cytoplasm. Cleavage and blastocyst rates were checked at 48 hpi and 6, 7, and 8 dpi, respectively. All expanded blastocysts were fixed in 0.5% glutaraldehyde (v/v) and stored at 4°C for TUNEL analysis and cell-number evaluation. Determination of Nuclear Maturation Stage Upon maturation, oocytes were washed in PBS-PVA, denuded from cumulus cells by gentle pipetting, fixed in 0.5% glutaraldehyde (v/v) for 15 min and stored at 4°C. The day of the analysis, oocytes were placed in a glass slide with 1 µL drop of Slowfade™ and 5 µg/mL Hoechst 33342 under a coverslip. After 20 min at room temperature, chromatin configurations were analyzed at 20X augmentation by fluorescence microscopy (Eclipse 80i, Nikon Instruments Europe, Amsterdam, The Netherlands). Oocytes showing a germinal vesicle (GV) chromatin configuration were considered immature, and those showing a metaphase plate and a polar body were categorized as matured metaphase II (MII) oocytes. Viability, Early Apoptosis, and Mortality Assessment Following the manufacturers' protocol, early apoptosis was assessed using Annexin V staining (Invitrogen©, Thermo Fisher Scientific, Barcelona, Spain). Denuded oocytes were placed in 100 µL Annexin V binding buffer droplets containing 5 µL of Annexin V/FITC and 1 µL of propidium iodide (PI; 100 µg/mL) and incubated at 37°C on a heated plate in the dark for 15 min. After washing three times in PBS-PVA, oocytes were mounted on slides in a 1 µL drop of Slowfade™ and 5 µg/mL Hoechst 33342. Oocyte nuclei were observed at 20X augmentation with an epifluorescence microscope (Eclipse 80i, Nikon Instruments Europe, Amsterdam, The Netherlands). Oocyte status was classified into the following categories: viable (Annexin V-/PI-), early apoptotic (Annexin V+/PI-), and dead (Annexin V-/PI + and Annexin V+/PI+). Representative images of different categories are shown in the Fig. 2 . Measurement of Reactive Oxygen Species (ROS) and Reduced Glutathione (GSH) Mature oocytes were incubated in 10 µM CM-H 2 DCFDA (Thermo Fisher Scientific, Barcelona, Spain) and 10 µM Cell Tracker Blue (Thermo Fisher Scientific, Barcelona, Spain) for 30 min at 37°C in the dark to detect intracellular reactive oxygen species (ROS) and reduced glutathione (GSH), respectively. The oocytes were then washed thrice in PBS-PVA and placed in slides for evaluation. Fluorescence intensity was observed at 20X augmentation using epifluorescence microscopy (Eclipse 80i, Nikon Instruments Europe, Amsterdam, The Netherlands) and the signal was quantified using ImageJ 1.45s software (National Institutes of Health, Bethesda, USA). Representative images of ROS and GSH levels in sheep oocytes are shown in the Fig. 3 . Mitochondrial Membrane Potential Analysis Membrane potential was determined by incubating ocytes for 30 min at 37°C in 0.5 µM of JC-1 dye (Thermo Fisher Scientific, Barcelona, Spain). After incubation, oocytes were washed twice for 5 min and then placed on glass slides. Oocytes were examined by 20X augmentation by fluorescence microscopy (Eclipse 80i, Nikon Instruments Europe, Amsterdam, Netherlands). Relative mitochondrial membrane potential was determined as the ratio of J-aggregate to J-monomer staining intensity with ImageJ 1.45s software (National Institutes of Health, Bethesda, USA). Representative images of JC-1 stained mitochondria in sheep oocytes is shown in the Fig. 4 . Assessment of Mitochondrial Distribution To determine mitochondrial distribution patterns, oocytes were subjected to double staining with MitoTracker© Red CMXRos (Thermo Fisher, Barcelona, Spain), a mitochondrial-specific probe and Hoechst 33342 to stain the chromosomes. Following IVM, oocytes were incubated for 20 min in PBS-PVA plus 100 nM MitoTracker© Red CMXRos at 37°C in the dark. The oocytes were washed thrice under agitation for 5 min and mounted in slides with 1 µL drop of Slowfade™ and 5 µg/mL Hoechst 33342. Oocytes were examined under 20X augmentation by fluorescence microscopy (Eclipse 80i, Nikon Instruments Europe, Amsterdam, Netherlands). Mitochondrial distribution was classified into two categories: abnormal mitochondrial distribution in the cytoplasm and normal distribution (Fig. 5 ). DNA Fragmentation Assay Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was used to detect DNA fragmentation in MII oocytes and expanded blastocysts. Samples were fixed in 4% glutaraldehyde for 15 min and permeabilized in 0.5% Triton X-100 for 1 h at room temperature. After, the In Situ Cell Death Detection kit (Merck Life Sciences, Madrid, Spain) was used to detect DNA strand breaks following the manufacturers’ instructions. Briefly, oocytes and blastocysts were placed in 30 µL drops of TUNEL reagent with deoxyuridine 5-trisphosphate (dUTP)-conjugated isothiocyanate fluorescein and incubated for 1 h at 37°C. The positive control was pre-incubated with DNAse (0.2 U/µL) for 1 h at 37°C, while the negative control was incubated in the absence of deoxynucleotidyl transferase enzyme. After that, samples were washed thrice in PBS-PVA and placed in slides in a 1 µL drop of Slowfade™ with 5 µg/mL Hoechst 33342. Samples were evaluated at 20X magnification by epifluorescence microscopy (Eclipse 80i, Nikon Instruments Europe, Amsterdam, The Netherlands). Oocytes and blastomeres with DNA damage, e.g., with a fragmented nucleus, were classified as TUNEL-positive and those without damage as TUNEL-negative (Fig. 6 ). Meiotic Spindle Configuration Assessment Mature oocytes were denuded and fixed with methanol (1:1) in PBS for 20 min at room temperature and stored in PBS-PVA at 4° C until use. After, oocytes were incubated in a permeabilizing solution (0.5% Triton-X-100 in PBS-PVA) for 30 min at room temperature, washed twice in PBS-PVA, and then blocked with 2% FCS in PBS for 45 min. Microtubules were detected using anti-α-tubulin (1:300) at 4°C overnight. After rinsing twice with 2% FCS in PBS for 5 min per wash, samples were incubated for 1 h at room temperature with secondary anti-mouse fluorescein isothiocyanate (FITC)–labeled secondary antibody (1:300) and Alexa Fluor 488-labelled anti-mouse IgG antibodies (Molecular Probes, Eugene, OR, USA; 1:300) at room temperature for 30 min. Oocytes were mounted on a glass slide in a drop of Slowfade with 5 µg/mL Hoechst 33342 to visualize chromosomes. Oocytes were examined at 20X augmentation by fluorescence microscopy (Nikon Eclipse 80i). Oocytes with a classical symmetric barrel-shaped spindle with chromosomes aligned regularly in a compact group along the equatorial plane were considered normal. In contrast, oocytes with spindles that were disorganized, clumped, dispersed, or missing (entirely or partially) with the aberration of chromatin arrangement, clumping, or dispersal from the spindle center were considered abnormal (Fig. 7 ). Flow Cytometry Analysis of Cumulus Cells Cumulus cells were collected from mature COCs and examined using a FlowSight® Imaging Flow Cytometer (Amnis, Merck-Millipore, Germany) as previously described [28]. Briefly, samples were stained with 10 µM YO-PRO-1 and 0.5 µM PI to study viability, apoptosis, and mortality. Viable cells were recorded as YO-PRO-1-/PI-, while YO-PRO-1+/PI- were deemed apoptotic. Cells stained with PI were considered dead. For mitochondrial activity, cells were incubated with 200 mM of MitoTracker™ Deep Red (Thermo Fisher Scientific, Barcelona, Spain) for 20 min at 38.5°C in the dark and then stained with 10 µM YO-PRO-1 and 0.5 µM PI. Viable cells with active mitochondria were considered as MitoTracker+/YO-PRO-1-. To study ROS and GSH intracellular levels in viable cells, samples were incubated with 10 µM of Cell Tracker™ Blue (Thermo Fisher Scientific, Barcelona, Spain) and 10 µM of CM-H 2 DCFDA (Thermo Fisher Scientific, Barcelona, Spain) for 30 min at 38.5°C followed by 0.5 µM PI staining. A compensation overlap was performed before each experiment, and 1000 events were acquired per sample. The raw data were analyzed using IDEAS® software (AMNIS), and out-of-focus cells, debris, and cell clumps were excluded from the analysis. Statistical Analysis Statistical analyses were performed using the IBM SPSS 24.0 (IBM Corp.; Armonk, NY, USA) software. Data were tested for normal distribution (Kolmogorov–Smirnov, and Shapiro–Wilk tests) and homogeneity of variances (Levene test). First, maximum temperature, maximum relative humidity and THI values were analyzed by factorial ANOVA followed by Bonferroni post hoc test considering type of season (winter, spring, summer, and autumn) as the fixed effect. In experiment 1, oocyte viability, early apoptosis, mortality, oxidative status, mitochondrial membrane potential and distribution, DNA fragmentation, maturation, fertilization, and embryo development rates, blastocyst quality and cumulus cells activity were analyzed by factorial ANOVA followed by Bonferroni post hoc test. For that, type of season was considered the fixed effect. In experiment 2, oocyte viability, early apoptosis, mortality, oxidative status, mitochondrial membrane potential and distribution, DNA fragmentation; maturation rates, meiotic spindle and chromosome organization; fertilization rates and embryo production and cumulus cells parameters were also analyzed by factorial ANOVA followed by Bonferroni post hoc test. For that, ovary storage temperature (30°C, 38.5°C, 40°C and 41°C) and the replicate were considered fixed effects. Differences with probabilities of p ≤ 0.05 were considered significant, and results are presented as mean ± SEM. Results Temperature Humidity Indices as Indicators to Heat Stress of Climatic Conditions The range in maximum temperature, maximum relative humidity and THI values for each season between 2019 and 2020 is illustrated in Table 1 . THI was significantly greater ( p < 0.05) in summer compared to winter and spring, although there was no difference with autumn. EXPERIMENT 1: Effect of season on oocyte and cumulus cells quality parameters After IVM, the collection of COCs during summer resulted in reduced ( p < 0.05) oocyte viability (29.37 ± 14.07%) compared to winter and autumn (84.44 ± 9.38 and 84.58 ± 11.49%, respectively), and a higher number ( p < 0.05) of apoptotic oocytes (67.50 ± 8.14%) in comparison to the rest of seasons (winter = 10.00 ± 7.18%; spring = 16.67 ± 7.18%; and autumn = 11.67 ± 8.79%; Fig. 8 A). The percentage of dead oocytes was similar between the different seasons ( p > 0.05). To determine the effect of season on ovine oocyte oxidative status, intracellular ROS and GSH were determined in the corresponding post-IVM oocytes. The levels of ROS were significantly higher ( p < 0.05) in the oocytes collected during autumn (145.97 ± 20.44) than in the spring (30.38 ± 16.69). Additionally, GSH was significantly greater ( p < 0.05) in autumn (142.76 ± 15.45) compared to the rest of seasons (winter = 85.17 ± 12.62; spring = 51.55 ± 12.62; and summer = 70.01 ± 14.31; Fig. 8 B). Both the normal distribution of mitochondria throughout the oocyte and mitochondrial membrane potential was significantly lower ( p < 0.05) in summer (67.14 ± 7.36% and 0.23 ± 0.04, respectively) compared to winter (96.67 ± 6.49%) in the former and to autumn (0.44 ± 0.04) in the latter (Fig. 8 C, 8 D), although there were no differences with the other seasons. The number of oocytes with fragmented DNA was higher during summer (25.92 ± 8.15%) than the rest of seasons (winter = 11.42 ± 7.19%; spring = 1.22 ± 7.19%; and autumn = 0 ± 8.80%) although the difference was not statistically significant ( p > 0.05). As shown in Table 2 , cumulus cells live/death status was significantly ( p < 0.05) reduced during summer compared to spring since a lower number of viable cells and a higher number of dead cells were found between these two seasons, although differences were no found between summer and winter and autumn. We also assessed cumulus cells' apoptosis, number of active mitochondria, and intracellular ROS and GSH content. In all parameters, oocytes showed similar values ( p > 0.05) throughout the whole year (Table 2 ). Effect of season on subsequent maturation and developmental competence of sheep oocytes Results did not show significant differences ( p > 0.05) in the maturation and fertilization rates (Table 3 ). Although the oocytes collected throughout the four seasons had similar cleavage rates ( p > 0.05), the percentage of blastocysts from the initial number of oocytes was significantly increased ( p < 0.05) in winter compared to summer and autumn (Table 3 ). In addition, the number of blastocysts from the cleaved embryos at 48 hpi was also significantly increased ( p < 0.05) during winter and spring compared to summer and autumn (Table 3 ). In vitro blastocysts produced during summer show reduced quality Although the number of cells of in vitro produced blastocysts was similar ( p > 0.05) among all seasons (winter = 122.02 ± 8.69%; spring = 127.11 ± 7.34%; summer = 117.61 ± 7.93%; and autumn = 104.11 ± 8.69%), the percentage of blastomeres showing DNA fragmentation was higher during summer (19.25 ± 2.71%) compared to winter (6.49 ± 2.97%) and spring (8.47 ± 2.51%; Fig. 9 ) although there was no difference with autumn. EXPERIMENT 2: Increasing the ovary storage temperature in an in vitro ovary storage model impairs the quality of sheep oocytes and cumulus cells As shown in Fig. 10 A, the results revealed that the storage of slaughterhouse sheep ovaries for 3 h at 30°C produced greater ( p < 0.05) oocyte viability values (73.44 ± 6.48%) in comparison with 40°C (34.23 ± 8.29%) and 41°C (16.48 ± 2.29%; Fig. 10 A). Additionally, the lowest ( p < 0.05) percentages of dead oocytes were observed at 30°C (9.37 ± 5.84%) and 38.5°C (29.68 ± 5.84%) in contrast to 41°C (69.55 ± 8.05%; Fig. 10 A). Intracellular ROS levels were higher at 30°C (91.20 ± 10.99) although there was no difference ( p > 0.05) with 38.5°C, 40°C and 41°C (Fig. 10 B). Moreover, the values for the GSH content were similar between temperatures (30°C = 95.64 ± 12.41; 38.5°C = 65.33 ± 12.41; 40°C = 44.68 ± 17.10 and 41°C = 86.57 ± 17.10; Fig. 10 B). Regarding the mitochondria, although the membrane potential in terms of fluorescence intensity did not show significant differences ( p > 0.05) among temperatures (30°C = 0.38 ± 0.02; 38.5°C = 0.36 ± 0.02; 40°C = 0.33 ± 0.03 and 41°C = 0.35 ± 0.03), the normal distribution of these organelles was higher at 30°C and 38.5°C (87.5 ± 6.43% and 82.68 ± 6.43%, respectively) than at 41°C (39.02 ± 8.87%; Fig. 10 C). The number of oocytes with fragmented DNA as measured by the TUNEL assay was significantly lower ( p < 0.05) after an ovary storage at 30°C (5.21 ± 7.83%) when compared to the rest of temperatures (38.5°C = 56.06 ± 7.83%; 40°C = 55.44 ± 10.79% and 41°C = 60.84 ± 10.79%; Fig. 10 D). As shown in Table 4 , the percentage of viable cumulus cells was significantly higher ( p < 0.05) at 30°C compared to 38.5°C and 40°C, although there was no different in relation to 41°C. The number of active mitochondria in cumulus cells was significantly greater ( p 0.05) between temperatures. Stage of the nuclear maturation, meiotic spindle, and chromosome organization with increasing ovary storage temperature The influence of temperature on oocyte nuclear maturation, cytoskeletal integrity, and chromosome organization after IVM was assesed. The rate of oocytes reaching MII phase was greater ( p < 0.05) in the 30°C group (69.82 ± 4.97%) compared to the rest of temperatures (38.5°C = 21.73 ± 4.67%; 40°C = 16.87 ± 6.43%; and 41°C = 9.82 ± 6.43%; Fig. 11 A). Given the significantly compromised maturation rates at 40°C and 41°C, the evaluation of meiotic spindle configuration was limited to the 30°C and 38.5°C groups. This limitation was necessary, as the low maturation rates at elevated temperatures made the reliable assessment of spindle integrity unfeasible. As expected, most of the 30°C group oocytes contained a typical MII spindle (91.24 ± 3.09%; Fig. 11 B). However, in the 38.5°C group a reduced ( p < 0.05) number of oocytes (49.41 ± 3.79%) presented a normal meiotic spindle configuration. Regardless, the proportion of oocytes with a normal organization of chromosomes was not statistically different ( p > 0.05) between the temperature’s groups (30°C = 90.96 ± 5.61%; and 38.5°C = 67.53 ± 6.88%; Fig. 11 B). Increasing ovary storage temperature reduces in vitro embryo production rates and blastocyst quality in sheep While no differences were observed in the fertilization rates ( p > 0.05), the results reported in Table 5 show that the oocytes collected from ovaries stored at 30°C yield cleavage rates higher ( p < 0.05) than at 38.5°C (only these 2 temperatures were studied because of the extremely low number of matured oocytes for 40°C and 41°C). Moreover, the number of expanded blastocysts from the total of oocytes in culture and the cleaved embryos at 48 hpi followed the same tendency and were significantly higher at 30°C compared with 38.5°C (Table 5 ). Although the blastocyst total cell number was similar ( p > 0.05) among temperatures, the use of 38.5°C increased ( p < 0.05) the percentage of blastomere DNA fragmentation compared to 30°C. Discussion The ever-growing global population is driving a notable increase in global food production 29 . Annual meat production, and particularly sheep meat demand in Europe, is expected to increase due to dietary diversification and demographic changes 2 , highlighting the growing importance of sheep and goat farming in the future 30 . At a time when increasing livestock production and productivity is key to meeting the escalating demand for animal protein globally, the climate is changing faster than the predictions, posing a significant challenge to the long-term viability of livestock production systems 31 . Mammals are able to maintain body temperatures higher than environmental (35–39°C) through a balance of body heat production and loss 32 and global warming can affect the proper functioning of that metabolism. Changes in ambient temperature over recent years and especially the increase in the global average surface temperature of about 0.6°C over the past 20th century can upset the balance 33 . Temperature has a clear effect on mammalian gamete's function, which shows high sensitivity to heat stress 34 in both in vivo and in vitro systems 35 . Extreme high-temperature conditions can increase body temperature and adversely impact mammalian biological functions, leading to impaired production and reproductive traits 36 , 37 . Most reproductive processes, including gametogenesis, fertilization or embryonic development, can be influenced by extreme high environmental temperatures that directly impact reproductive performance 31 , 38 , 39 . Even though sheep are found to be well adapted to different environmental conditions including high environmental temperatures 30 , heat stress significantly impairs reproduction and entails a risk to the efficiency of meat production 39 . To meet increasing global demands and minimize environmental consequences, advances in animal agriculture via reproductive technologies will be essential. These technologies aim to enhance livestock efficiency and productivity while adjusting to climate change and global warming 40 . Nowadays, assisted reproduction techniques are widely used in livestock, though IVP has been little explored because of its poor performance 41 . The collection of developmentally competent oocytes is a drawback that may limit IVP applicability. Several studies have demonstrated that heat stress adversely impacts on fertility process, especially in oogenesis, oocyte function, maturation, fertilization, and blastocyst development 42 . The molecular study of oocyte damage triggered by heat stress could help to avoid this type of injury. The present work compares, for the first time, whether the effects of heat stress season-dependent (winter, spring, summer, and autumn) are replicated in the in vitro model which is crucial for a deeper understanding of the physiological mechanisms underlying oocyte damage. Thus, we evaluate the evolution of oocyte competence, fertility and blastocyst rates throughout the seasons and the effects of high in vitro temperatures on oocyte quality and its competence for in vitro embryo production. Different authors have shown that the quality of the oocyte, and consequently the production of embryos, is affected by seasonality in small ruminants. Souza-Fabjan et al., 43 showed that blastocyst production in goats was higher during fall and lower in spring. On the other hand, Serra et al., 44 demonstrated a seasonal effect in sheep with better oocyte quality results in spring compared to autumn, although this study was carried out in prepubertal animals where the endocrine profile is quite different from adults 45 . In our case, the data generally showed a poorer oocyte quality for the summer months. We hypothesize that this fact was due to the high temperatures registered in our country at that time of the year. In this way, Table 1 shows that the level of heat stress reached during summer was considered to be severe (79–83) to ruminant species, while the rest of seasons showed values considered as normal (≤ 74). We think that the low quality shown during summer is not due to a photoperiod seasonal effect since some parameters analyzed showed better results during spring which is a non-optimal reproductive season for sheep species in our latitude. Therefore, what should have been expected during spring considering the photoperiod is a worse quality also at this time of the year. Apoptosis is a form of programmed cell death that plays a crucial role in maintaining the homeostasis of various biological processes 46 and plays an important role in the disruption of the normal function of oocytes under thermal stress 47 . In this study, Annexin-V staining has been used as an early marker of apoptosis, detecting alterations in the oocyte phospholipid membrane and preceding late stages of apoptosis assessed by TUNEL staining. The early apoptosis, assessed using the V-FITC assay, is more frequent in oocytes subjected to heat stress, suggesting that summer high temperatures can induce apoptosis in oocytes 48 . In fact, our results reported by Annexin-V staining showed that the percentage of viable oocytes decreases during summer, while the apoptotic oocyte rate increases. Likewise, Ahmadi 49 observed a significant interaction between season and thermal stress on apoptosis in sheep, where the reduced developmental competence observed in heat-stressed oocytes was partially linked to changes in their plasma membrane. Similarly, the percentage of viable oocytes decreased for higher in vitro temperatures (40°C and 41°C) in relation to 30°C. Our results are in agreement with previous studies performed in bovine where oocytes subjected to heat stress before 12 h maturation 48 , or even in the short-term at the early stage of maturation, resulted in increased Annexin-V binding and oocytes that undergo early apoptosis compared to the control group 50 . Heat stress has been shown to activate the apoptotic cascades, inducing alterations in the oocyte phospholipid membrane 51 and ultimately promoting a detrimental on oocyte developmental competence 49 . However, in our results the stress caused by the environmental temperature was not enough to damage oocyte DNA, although in vitro temperatures over 38.5°C dramatically damaged the oocyte DNA. The different effect observed between ambient heat stress and in vitro heat stress may be caused by the differences in the response of both models to high temperatures. Under physiological systems, provided the stress is not excessive, the organism's defense system will deal with potential injuries 52 . Thus, during the summer months, early apoptosis greatly increases unlike dead cell percentage or DNA damage. Early apoptosis is an event that can be reversed by the body's defense systems as the synthesis of antiapoptotic proteins or the synthesis of antioxidants 52 . Indeed, some studies have shown that heat shock induces early apoptosis events though upstream of DNA fragmentation 53 . This in vivo adaptational phenomenon was previously noted in bovine 54 and sheep 49 oocytes and even spermatozoa 55 . The development of heat tolerance could be associated with the expression of heat shock proteins, such as heat shock protein-70, which protects oocytes from apoptotic stimuli that harmfully affect DNA 56 . Thus, it would also be of interest to develop future experiments to determine if heat stress significantly impacts the expression levels of specific genes for both in vitro and in vivo models. Despite the result observed during the seasons, after excessive in vitro heat stress, the oocyte may not be able to counteract this damage as it may not have an active defense system, accumulating irreversible damage that leads to higher dead cell percentage and DNA damage. Actually, our findings are in line with other works where ovaries exposed to severe in vitro heat stress (high storage temperature and/or long-term storage) increased the number of oocytes with DNA fragmented nuclei 57 . Regarding the oxidative balance, our results have shown that GSH and ROS production was higher during autumn compared to the rest of the seasons for the former parameter and compared to spring for the latter as previously reported 44 . The balance between ROS production and antioxidant capacity also affects the developmental competence of oocytes. Under physiological conditions, ROS regulate specific cellular functions while high levels lead to various forms of cellular damage to DNA, proteins, lipids and ultimately affecting oocyte quality and viability 58 . Although an excess of ROS may lead to a harmful effect on oocytes, it has been shown that the presence of ROS derived from mitochondrial respiration is necessary for certain cell signaling pathways involved in folliculogenesis, oocyte maturation, embryogenesis and implantation 59 . In our latitude, the reproductive season in small ruminants begins from the end of summer to the beginning of winter. This fact drives us to think that during this period, higher cellular activity will be needed, the mitochondria will be more active, and more ROS will be generated. Additionally, our results are in line with other studies performed on sheep under similar latitudes, where oocytes collected during autumn also showed a significantly higher ROS production compared with other seasons 60 . Among the main antioxidants that protect the oocyte against oxidative damage, GSH plays a key role, showing the cytoplasmic maturity degree and the quality of the oocyte after in vitro maturation 61 . The regulation of intracellular redox potential in the oocyte is a crucial determinant of fertility and embryo development 62 , 63 . Thus, the high concentrations of GSH observed during autumn, compared with the rest of the seasons, could have been increased in order to achieve an oxidative balance and physiologically counteract the high production of ROS. Similarly, other works have also reported that animals may respond to temperature challenges by upregulating antioxidant enzymes 64 . Moreover, variation in antioxidant levels was suggested not only as a defense mechanism in order to counteract oxidative damage but also as a consequence of environmental conditions 65 . Regarding the effect of in vitro heat stress, the temperature increase did not affect the production of ROS and GSH. This may be due to the high percentage of dead oocytes that we found for temperatures over 38.5°C where the physiological system that determines the oxidation state of a cell could not be activated. Furthermore, we hypothesize that it is possible that live oocytes were unable to display the mechanisms that lead to the production of antioxidant enzymes due to the damage produced, hence this effect could not be visualized 66 . Concerning mitochondrial parameters, it is well known that a dysfunction in one or more aspects of mitochondrial biology results in reduced oocyte developmental competence. We observed that both mitochondrial activity and the homogeneous distribution of mitochondria were affected throughout the seasons. Moreover, this study shows not only heat stress-induced alterations in mitochondrial distribution 67 but also observes a trend where homogeneous mitochondrial distribution aligns with mitochondrial membrane potential during summer. Thus, mitochondrial activity was higher during the autumn season compared to spring and summer and the percentage of oocytes with a normal distribution of mitochondria was lower in summer than in winter. These results agree with other studies where mitochondrial distribution differs between cold and warm seasons 67 , 68 . Particularly, our findings are aligned with previous research indicating that mitochondria exhibit less uniform distribution during summer 69 or afterward severe in vitro heat exposure (41°C) 48 . This fact could be due to an alteration of the cytoskeletal proteins caused by heat, as has been previously demonstrated by Gendelman and Roth (2012). Extremely high temperatures have been shown to denature cytoskeletal proteins that leads to its abnormal distribution in the oocyte 70 , leading mitochondria to relocate to the cell periphery instead of maintaining a homogeneous distribution throughout the cytoplasm 71 . This alteration of the cytoskeletal elements can even lead to the disruption of nuclear maturation and the disassembly of the meiotic spindle 72 . It is well known that meiotic spindle microtubules are susceptible to temperature changes which can ultimately cause chromosomal imbalance and cell death 73 – 75 . The microtubular network in the oocyte is critical for meiotic spindle formation, chromosome segregation, fertilization, and embryonic development 44 . Microtubules and their post-translational modification, especially tyrosination and acetylation in sheep oocytes 76 , play a crucial role in oocyte maturation and fertilization due to their influence on meiotic spindle assembly and chromosome movement. A balance between tyrosination (more abundant in dynamic microtubules) and acetylation (characterizing stability) is essential for the normal function of the meiotic spindle, which requires both dynamism and stability. Nevertheless, temperature has been shown to disrupt this balance, potentially affecting oocyte developmental competence 44 . Several studies have demonstrated that in vitro heat stress has detrimental effects on various aspects of cytoplasmic and nuclear oocyte maturation 77 , including cytoskeletal rearrangement 74 , meiotic spindle formation 72 , 73 and early embryonic mitotic failures 78 . In the present study, we also observed that the meiotic spindle was affected by a short exposure for 3 hours at 38.5°C, resulting in a lower percentage of oocytes with a normal spindle configuration. This parameter had a clear association with the low maturation rates of high temperatures (from 38.5°C). However, oocyte maturation was not affected by the temperatures recorded throughout the seasons. Unfortunately, the spindle configuration could not be assessed in this experiment therefore no data are available on this parameter. Our findings are consistent with other studies conducted on bovine oocytes which reported no significant seasonal effects on nuclear maturation 54 , 79 and a normal uniform alignment of the chromosomes on the spindle 54 . Regarding the cleavage rate, there were no differences between seasons, although the blastocysts percentage was lower during the summer and autumn. The fact that oocytes obtained in the summer season showed no differences with those collected during the rest of the year in terms of nuclear maturation and cleavage rate could reflect that immature oocytes can be fertilized and undergo cleavage without developing to the blastocyst stage 39 . In addition, the embryos obtained during those months had a higher number of apoptotic cells compared to winter and autumn. Similar results were found by Gendelman and Roth (2012). Furthermore, genetic studies suggest that heat stress during the summer months impedes embryo development, likely due to altered expressions in these conditions associated with a reduced developmental capacity of oocytes and embryos 80 . On the other hand, storing the ovaries for 3 hours at temperatures above 38.5°C produced a lower percentage of cleavage and blastocysts compared to 30°C in alignment with previous studies 48 . This decrease is likely to be a consequence of hyperthermia. It is important to note that these blastocysts were obtained from oocytes subjected to heat stress, which was previously observed to affect their quality and developmental competence in sheep 39 . The nuclear maturation and spindle configuration were significantly impaired for oocytes exposed to in vitro heat stress compared to their unexposed counterparts. This impairment could potentially result in irreparable damage to the percentage of oocytes progressing past the cleavage stage, leading to a subsequent decline in embryo developmental potential 48 . Similarly, the number of apoptotic cells was higher for blastocysts from ovaries stored at 38.5°C. These results agree with those obtained by Gendelman and Roth (2012a, b) 70 , 81 . Cumulus cells play critical roles in the maturation of the nucleus and cytoplasm in the oocyte since they allow the interchange of molecules with the oocytes 82 . In addition, they support energy production in the COCs and protect against oxidative stress-induced apoptosis. It was demonstrated that CCs are also important for the thermal protection of oocytes, as they provide extracellular thermoprotective molecules through gap junctions 83 , 84 . In addition, CCs also produce regulatory molecules that activate thermoprotective mechanisms within the oocyte 85 . Nevertheless, heat stress has been reported to reduce the effectiveness of the gap junction communication between cumulus cells and oocytes 86 . As expected, we have reported a lower percentage of viable CCs during the summer season as well as a higher percentage of dead cells compared with other seasons. Nevertheless, apoptotic percentage, ROS/GSH levels and mitochondrial activity were not affected. On the other hand, a lower viable percentage for the temperature 38.5°C was observed in support of oocyte apoptosis assay results. In this line, different studies have seen that the apoptosis incidence in cumulus cells is negatively correlated to the developmental competence of oocytes and subsequent embryonic development after fertilization 87 – 89 . Nevertheless, other works showed that oocytes were more sensitive to heat stress than cumulus cells 90 and just a long exposure to heat stress conditions may overwhelm the cumulus cells protection capacity 91 . Moreover, although we have not studied morphological defects, cumulus morphology can also be affected by in vitro heat stress during ovaries storage 92 as well as during summer rather than in winter 93 . To the best of our knowledge, no studies have simultaneously evaluated the effects of collection season on oocyte quality and IVP in sheep while also monitoring the impact of varying ovarian storage temperatures on oocyte quality and competence. The results revealed that extreme environmental temperatures during summer significantly decreased oocyte and cumulus cells viability, DNA integrity and mitochondrial normal distribution. Moreover, ovary collections during summer had not only lower blastocyst percentage but also summer-produced blastocysts exhibited significantly higher DNA fragmentation compared to winter. This harmful effect was not limited to summer seasons since it was carried over to autumn, indicating a carry-over effect caused by heat stress during the summer. Similarly, the severity of the damages caused during in vitro heat stress was related to the highest temperatures selected, where temperatures over 38.5°C caused major injuries on oocyte quality parameters assessed and blastocyst rates. These findings clearly demonstrated that both seasonal and in vitro extreme temperatures significantly impaired oocyte quality and embryo production, emphasizing the need to develop strategies to reduce the impacts of heat stress and enhance reproductive outcomes in sheep. Declarations Data availability All data generated or analyzed during this study are included in this article. Authors’ contributions Conceptualization: AJS.; methodology: AM-M, IS-A, MI-C, D-AM-C, CM and RF-S; formal analysis: AM-M and IS-A; resources: AJS and JJG.; writing: AM-M, IS-A and AJS; original draft preparation: AM-M; writing—review and editing: AJS and JJG; funding acquisition: AJS and JJG. All authors have read and agreed to the published version of the manuscript. Funding This study was funded by the Spanish Ministry of Economy and Competitiveness (AGL2017-89017-R). AM-M. was supported by a Ministry of Economy and Competitiveness scholarship (PRE2018-084837). 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Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Published Journal Publication published 17 May, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Feb, 2025 Reviews received at journal 31 Jan, 2025 Reviews received at journal 24 Jan, 2025 Reviewers agreed at journal 11 Jan, 2025 Reviewers agreed at journal 09 Jan, 2025 Reviews received at journal 05 Jan, 2025 Reviewers agreed at journal 26 Dec, 2024 Reviewers invited by journal 21 Dec, 2024 Editor assigned by journal 21 Dec, 2024 Editor invited by journal 19 Dec, 2024 Submission checks completed at journal 18 Dec, 2024 First submitted to journal 06 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5595754","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":391886428,"identity":"8d41d2d5-46f1-42b0-8c0d-f3565c609d2a","order_by":0,"name":"Alicia Martin-Maestro","email":"","orcid":"","institution":"University of Castilla-La Mancha","correspondingAuthor":false,"prefix":"","firstName":"Alicia","middleName":"","lastName":"Martin-Maestro","suffix":""},{"id":391886429,"identity":"e76ea6d1-5219-4d2e-be8d-26e54f52154a","order_by":1,"name":"Irene Sánchez-Ajofrin","email":"","orcid":"","institution":"University of Castilla-La Mancha","correspondingAuthor":false,"prefix":"","firstName":"Irene","middleName":"","lastName":"Sánchez-Ajofrin","suffix":""},{"id":391886430,"identity":"d20162ad-1000-4cec-bdb9-f22226b4ed95","order_by":2,"name":"María Iniesta-Cuerda","email":"","orcid":"","institution":"University of Castilla-La Mancha","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"","lastName":"Iniesta-Cuerda","suffix":""},{"id":391886431,"identity":"d4321a16-3bf9-428e-a245-f5c56772a81c","order_by":3,"name":"Daniela Medina-Chávez","email":"","orcid":"","institution":"University of Castilla-La Mancha","correspondingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Medina-Chávez","suffix":""},{"id":391886432,"identity":"9665bcf3-d814-4bd1-acdb-c2ebef5d0d8c","order_by":4,"name":"Carolina Maside","email":"","orcid":"","institution":"University of Castilla-La Mancha","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Maside","suffix":""},{"id":391886433,"identity":"c2398569-05cf-417c-ae02-43c101a39bf7","order_by":5,"name":"María Fernández-Santos","email":"","orcid":"","institution":"University of Castilla-La Mancha","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"","lastName":"Fernández-Santos","suffix":""},{"id":391886434,"identity":"d8c37a2c-c428-4b95-930a-2efffa32c5a1","order_by":6,"name":"Julián Garde","email":"","orcid":"","institution":"University of Castilla-La Mancha","correspondingAuthor":false,"prefix":"","firstName":"Julián","middleName":"","lastName":"Garde","suffix":""},{"id":391886435,"identity":"d879522e-74e4-4755-b1bc-88a40dfb36c7","order_by":7,"name":"Ana Josefa Soler","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIie2PMWrDMBSGXwg4i1uvr4t9hWcEnUrP8kShXhIIdMkQgqDUPUPoJTx1VjHYiw+goRRn6awDmLSKu7SDko6F6kNCAr0P/T9AIPBHIbeT8dYDpABT9SvlYhxjAAEwOa3Ad0WqU0rydN8v7XCFiZrtel69FVX78lDBauNV8LWhfFveIupYEHd3i6qTpYGu9n9jmMSZqjeg4whlyYvKTEq3tNfITGFFPHxgpmfvKPdc0Kjs/cHIzEnEkUbScIlSMX8paupVcjNfui43mNeHLg3n20MXbvxdUlM8kx2uMW0fd71dc3be1o2xa38wR0Tj8SMJHxPcbH/8PRAIBP49n0z6WsAmwMbsAAAAAElFTkSuQmCC","orcid":"","institution":"University of Castilla-La Mancha","correspondingAuthor":true,"prefix":"","firstName":"Ana","middleName":"Josefa","lastName":"Soler","suffix":""}],"badges":[],"createdAt":"2024-12-06 19:23:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5595754/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5595754/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-01173-1","type":"published","date":"2025-05-17T15:57:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":72034529,"identity":"75d37998-fc9c-48bb-a20e-4bcd73b1efc9","added_by":"auto","created_at":"2024-12-20 22:47:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":243458,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSchematic representation of experimental design.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5595754/v1/d726495aef647c3251ba87d6.png"},{"id":72034526,"identity":"5af3f614-4f4a-4b93-abe5-f1e603be70c9","added_by":"auto","created_at":"2024-12-20 22:47:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":260808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRepresentative images of early apoptosis detection in sheep oocytes. A) Early apoptotic oocytes: Annexin V positive represented by green signal across the oocyte membrane. B) Viable oocytes: no green Annexin V and red PI signal. C-D) Dead cells: PI positive signal. Scale bar = 50 µM.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5595754/v1/41c6acab07cb3094993dd869.png"},{"id":72034841,"identity":"85703c5f-9f96-4860-b1a6-104a1aa79e79","added_by":"auto","created_at":"2024-12-20 23:03:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":47866,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRepresentative images of reactive oxygen species (ROS) and glutathione (GSH) levels in sheep oocytes. (A) High ROS and GSH intensity. (B) Low ROS and GSH intensity. Scale bar = 50 µM.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5595754/v1/6f386d81e3667b96e20202d0.png"},{"id":72034843,"identity":"73d6ec45-d4c0-486a-8d17-7340956c4151","added_by":"auto","created_at":"2024-12-20 23:03:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":17889,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRepresentative images of JC-1 stained mitochondria in sheep oocytes. (A) mitochondria with low membrane potential (green fluorescence). (B) Mitochondria with high membrane potential (red fluorescence). Scale bar = 50 µM.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5595754/v1/ca879b61452985731c349ff0.png"},{"id":72034532,"identity":"95f6f7f2-5efc-451b-a6e8-a450da1a20ba","added_by":"auto","created_at":"2024-12-20 22:47:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":20235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMitochondrial distribution classification. A)\u003c/em\u003e Normal d\u003cem\u003eistribution. B) Abnormal mitochondria distribution. Scale bar = 50 µm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-5595754/v1/b76c3a298723f60f2816de31.png"},{"id":72034535,"identity":"b079fca2-a937-4724-8b2f-2a6a66845fe5","added_by":"auto","created_at":"2024-12-20 22:47:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":72225,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTUNEL staining in oocytes (A and B) and blastocysts (C, D and E). A) TUNEL positive oocyte (green/yellow nucleus). B) TUNEL negative oocyte (red nucleus). Scale bar = 50 µm. C) Hoechst 3342 staining. D) Blastocyst with TUNEL positive cells in green. E) Merge. Scale bar = 30 µm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-5595754/v1/8ff968ebea2ced12c0fd3121.png"},{"id":72034531,"identity":"9da4a2a9-5b5f-43e2-b523-af0abe4a0899","added_by":"auto","created_at":"2024-12-20 22:47:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":393662,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRepresentative images of spindle (green) morphology and chromatin (blue) alignment in ovine oocytes following immunostaining. Noted that normal morphology shows symmetrical barrel shape meiotic spindle with chromatin aligned regularly along the equatorial plane of spindle, while abnormal illustrates reduced spindle, absent spindle or asymmetrical spindles and chromatin. Scale bar=50 μm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-5595754/v1/3063b8f98e853bdbb87d8c47.png"},{"id":72034544,"identity":"29d3c2c5-279a-4322-af2d-a80919046c5a","added_by":"auto","created_at":"2024-12-20 22:47:07","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":93096,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eThe effect of season on oocyte quality parameters in sheep. (A) Viability and early apoptosis rates; (B) ROS and GSH levels; (C) Mitochondrial membrane potential: red (high membrane potential)/green (low membrane potential) fluorescence ratio (JC-1 staining); (D) Normal mitochondrial distribution. Results are expressed as mean ± SEM. \u003c/em\u003e\u003csup\u003e\u003cem\u003ea,b\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Different letters indicate differences between seasons for each parameter.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-5595754/v1/e9151a960d7f2bb28829ac4c.png"},{"id":72034543,"identity":"67f2c771-d85e-49e0-8de4-ab6eeba1e498","added_by":"auto","created_at":"2024-12-20 22:47:07","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":24420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eThe effect of season on the DNA fragmentation in sheep blastocyst. Results are expressed as mean ± SEM. \u003c/em\u003e\u003csup\u003e\u003cem\u003ea,b\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Different letters indicate differences between seasons.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-5595754/v1/d6d63eccfe53c60f32f21a42.png"},{"id":72034540,"identity":"acbec9a5-0872-4cdb-bcd9-bc9362bcfc75","added_by":"auto","created_at":"2024-12-20 22:47:07","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":54227,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eThe effect of in vitro heat stress during ovary storage on oocyte quality parameters in sheep. (A) Viability and early apoptosis rates; (B) ROS and GSH levels; (C) Normal mitochondrial distribution; (D) DNA fragmentation. Results are expressed as mean ± SEM. \u003c/em\u003e\u003csup\u003e\u003cem\u003ea,b\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Different letters indicate differences between temperatures for each parameter.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-5595754/v1/7a283d7915fb1986a428cacf.png"},{"id":72034730,"identity":"f5dac53e-5663-4085-8790-99e37a472044","added_by":"auto","created_at":"2024-12-20 22:55:07","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":21380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eThe effect of in vitro heat stress during ovary storage on oocyte nuclear maturation stage, spindle and chromosomal organization after IVM in sheep. Results are expressed as mean ± SEM. \u003c/em\u003e\u003csup\u003e\u003cem\u003ea,b\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Different letters indicate differences between temperatures for each parameter.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage16.png","url":"https://assets-eu.researchsquare.com/files/rs-5595754/v1/150d70b379ad5c95fd12f96f.png"},{"id":83067822,"identity":"dc05aae5-d85d-44ee-b632-596161fa1968","added_by":"auto","created_at":"2025-05-19 16:06:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2633041,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5595754/v1/39f63735-4135-431f-bb6e-48b926bad320.pdf"},{"id":72034522,"identity":"e00fc837-499b-4574-a656-e45cf6dff1bf","added_by":"auto","created_at":"2024-12-20 22:47:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":410939,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5595754/v1/2c614f377048e1c6e9df7e6f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"How Heat Stress Affects the Functionality of the Ovine Cumulus-Oocyte Complex: Implications for In Vitro Embryo Production","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDriven by growth in developing countries, the world population is projected to reach 8.5 and 9.7\u0026nbsp;billion people by 2030 and 2050, respectively, thereby requiring around a 70% increase in food production globally \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Annual meat production, for instance, would have to grow by over 200\u0026nbsp;million tons \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Particularly in Europe, sheep meat is also expected to increase due to the diversification of meat diets and changes in the population structure \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. To meet these demands and reduce environmental impact, advances in animal agriculture through reproductive biotechnologies will be necessary to provide increasingly efficient and productive livestock and adapt to climate change and global warming \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In this context, the application of advanced assisted reproductive technologies, such as \u003cem\u003ein vitro\u003c/em\u003e embryo production (IVP), in the farming sheep industry is highly promising and offers several advantages since it allows the production of embryos from oocytes recovered from unstimulated ovaries, non-fertile females, prepubertal, pregnant, senile, and even dead or slaughtered animals \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In addition, IVP facilitates the preservation of valuable biospecimens and provides secure transportation of biological material. Nevertheless, despite substantial improvements, the efficiency and quality of IVP-derived embryos are still one of the main challenges of livestock farming \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGlobally, new research into how climatic conditions heavily affect reproduction in domesticated farm animals is gaining interest throughout the agricultural sector \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. From the standpoint of economic efficiency, the reproductive ability of domestic food animals such as sheep is the most critical trait that may be compromised by climate stressors \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Previously, fundamental research into environmental stress during summer and its influence on sheep has provided evidence that heat stress can negatively influence oocyte developmental competence and result in embryonic loss \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Heat stress may lead to changes in oocyte membrane properties such as decreased phospholipid polyunsaturated fatty acid content \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, which are essential for gamete fertility \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Furthermore, it may alter the transcript levels of genes involved in oogenesis, folliculogenesis, and embryonic development \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and impair nuclear and cytoplasmic maturation events such as translocation of cortical granules and cytoskeletal rearrangement \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e that can eventually lead to apoptosis \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In addition, heat stress may affect the spatial distribution of mitochondria within the oocyte, possibly due to alterations in the cytoskeleton \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, as well as the proportion of highly polarized mitochondria and the expression of developmentally important genes \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMoreover, in summer, it is not unusual for sheep IVP systems to endure some periodic reductions in embryo yield \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In fact, most laboratories cease their activity during the hottest months. Previously, variability in ovine IVP output throughout the year was attributed to an effect of season, related to photoperiod \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, among the several variables that influence seasonal patterns of blastocyst production is also environmental temperature \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Consequently, understanding the effects of high temperatures that prevail throughout the warm season on oocyte quality may be a prerequisite for the successful implementation of IVP \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTherefore, in the present study, the first objective was conducted to evaluate the effect of the season (periods with differences in ambient temperature and humidity) in which the sheep ovaries are harvested (winter, spring, summer, and autumn) on oocyte quality and \u003cem\u003ein vitro\u003c/em\u003e embryo production. Additionally, we have examined the impact of heat stress through elevated ovarian temperature in an \u003cem\u003ein vitro\u003c/em\u003e model to understand better the physiological mechanisms of oocyte damage. Such research may ultimately help develop novel strategies to mitigate the impact of summer heat stress on oocyte quality and protect the integrity of the female germline in small ruminants.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eAll chemicals were acquired from Merck Life Sciences (Madrid, Spain) unless stated otherwise.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Design\u003c/h2\u003e \u003cp\u003eIn the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e is shown the experimental design. In experiment 1, to study the effect of season on oocyte quality and developmental competence, adult sheep ovaries were collected twice a month from an abattoir located in south-eastern Spain (Murcia; latitude: 37\u0026deg; 59' 13.34\" N; longitude: -1\u0026deg; 07' 48.14\" W) receiving sheep from surrounding areas during winter (December-February), spring (March-May), summer (June-August), and autumn (September-November) and transported to the laboratory within 3 h at 30\u0026deg;C in physiological saline (8.9 gr/L NaCl) supplemented with penicillin (0.1 g/L). Immediately after arrival, ovaries were processed. To study maturation rates and oocyte quality parameters, 1049 cumulus-oocyte complexes (COCs) were \u003cem\u003ein vitro\u003c/em\u003e matured and further examined. The remaining 1642 COCs were subjected to \u003cem\u003ein vitro\u003c/em\u003e fertilization (IVF) and \u003cem\u003ein vitro\u003c/em\u003e culture (IVC) to evaluate fertilization potential, embryo development, and blastocyst quality.\u003c/p\u003e \u003cp\u003eIn experiment 2, to investigate the effect of induced heat stress on oocyte quality and developmental competence, an \u003cem\u003ein vitro model\u003c/em\u003e was developed. For this purpose, adult sheep slaughterhouse ovaries were collected post-mortem from the same abattoir as experiment 1 receiving sheep from surrounding areas during late autumn and winter. Afterwards, ovaries were transported in physiological saline (8.9 gr/L NaCl) supplemented with penicillin (0.1 g/L) at 30\u0026deg;C, 38.5\u0026deg;C, 40\u0026deg;C and 41\u0026deg;C and stored for 3 h before processing. Then, the quality and developmental potential of 1509 oocytes was evaluated after \u003cem\u003ein vitro\u003c/em\u003e maturation (IVM).\u003c/p\u003e \u003cp\u003eAll experiments of collection and evaluation of COCs, in vitro maturation and fertilization of oocytes, assessment of fertilized oocytes, embryos and cumulus cells were conducted following the procedures previously described by our working group \u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eClimate Data and Calculation of Temperature Humidity Index for Heat Stress Assessment\u003c/h3\u003e\n\u003cp\u003eDaily observed meteorological data for 2019 to 2020 (time of ovarian collection) on each ovary collection season were obtained from the State Meteorological Agency of Spain for the location of Puerto Tocinos (Murcia). Daily values for maximum temperature measurements and relative humidity (RH hereafter) were used to determine mean values for each variable per season. As previously described by Caraba\u0026ntilde;o et al. \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, the temperature humidity index (THI) was used to assess the potential for heat stress on sheep at our latitude. The THI was formulated specifically to ruminant species \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and was examined for seasonal patterns of variability. The THI formula used is shown below, with temperatures in degrees Celsius and RH expressed as percentage:\u003c/p\u003e \u003cp\u003eTHI ruminant = (1.8 T\u003csub\u003emax\u003c/sub\u003e + 32)\u0026ndash;((0.55\u0026ndash;0.0055 RH) (1.8 T\u003csub\u003emax\u003c/sub\u003e\u0026ndash;26.8))\u003c/p\u003e \u003cp\u003eThe level of heat stress was considered as: normal\u0026thinsp;\u0026le;\u0026thinsp;74; moderate 75\u0026ndash;78; severe 79\u0026ndash;83; very severe (emergency)\u0026thinsp;\u0026ge;\u0026thinsp;84 \u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eOocyte Collection and In Vitro Maturation\u003c/h3\u003e\n\u003cp\u003eImmature COCs were retrieved from the follicles using a scalpel blade in 2 mL of collection medium (TCM199 medium supplemented with 2.38 mg/mL HEPES, 2 \u0026micro;L/mL heparin, and 4 \u0026micro;L/mL gentamicin). Immediately, the COCs with clear or moderately granular ooplasm surrounded by at least three layers of packed cumulus cells were selected and homogeneously distributed in selection media (TCM199 medium supplemented with 2.38 mg/mL HEPES and 4 \u0026micro;L/mL gentamicin). Then, the COCs were washed in TCM199 and 4\u0026micro;L/mL gentamicin. Subsequently, COCs were homogeneously distributed in 4-well plates with 500 \u0026micro;L of maturation medium: TCM199 and 4 \u0026micro;L/mL gentamicin, 100 \u0026micro;M cysteamine, 10 \u0026micro;g/mL follicle-stimulating hormone (FSH), 10 \u0026micro;g/mL luteinizing hormone (LH), and 10% fetal calf serum (FCS). The maturation medium was covered in mineral oil (Nidacon, Gothenburg, Sweden), and COCs were incubated for 24 h at 38.5\u0026deg;C, 5% CO\u003csub\u003e2,\u003c/sub\u003e and maximal humidity.\u003c/p\u003e\n\u003ch3\u003eIn Vitro Fertilization\u003c/h3\u003e\n\u003cp\u003eGroups of approximately 40\u0026ndash;45 mature oocytes were placed in four-well dishes containing 500 \u0026micro;L of fertilization medium: synthetic oviductal fluid (SOF; \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e) supplemented with 10% estrous sheep serum (ESS). Oocytes were subjected to IVF using frozen semen of two rams from the germplasm bank of the \"Biology of Reproduction Group\" of the Universidad de Castilla-La Mancha (UCLM), which is authorized for the collection and storage of sheep semen (ES008007). Thawed spermatozoa were separated using Percoll\u0026copy; density gradient (45%/90%) and capacitated for 15 min at 38.5\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e in fertilization medium. Then, spermatozoa (1 x 10\u003csup\u003e6\u003c/sup\u003e/mL) and oocytes were co-incubated at 38.5\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e and maximal humidity.\u003c/p\u003e\n\u003ch3\u003eIn Vitro Embryo Culture\u003c/h3\u003e\n\u003cp\u003eAfter 18 h post-insemination (hpi), putative zygotes were washed by repeated pipetting and transferred to 25 \u0026micro;L drops (about one embryo per \u0026micro;L) of culture medium (SOF supplemented with 3 mg/mL bovine serum albumin), covered with mineral oil and cultured until day 8 post-insemination (dpi) at 38.5\u0026deg;C in a humidified atmosphere and 5% CO\u003csub\u003e2\u003c/sub\u003e, 5% O\u003csub\u003e2\u003c/sub\u003e and 90% N\u003csub\u003e2\u003c/sub\u003e in air.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of Fertilization and Embryo Production Rates\u003c/h2\u003e \u003cp\u003eAfter IVF, oocytes were fixed in 0.5% glutaraldehyde (v/v) for 15 min at room temperature and stored at 4\u0026deg;C until analysis. To examine sperm penetration, cells were stained with Hoechst 33342 (5 \u0026micro;g/mL) for 20 min at room temperature, washed in phosphate-buffered saline (PBS) supplemented with 0.1% PVA (w/v; PBS-PVA), and then analyzed with 20X augmentation by fluorescence microscopy (Eclipse 80i, Nikon Instruments Europe, Amsterdam, Netherlands). Oocytes containing both female and male pronuclei (regardless of the stage of decondensation) relative to the total number of oocytes matured were considered to be fertilized and were classified as normal (2PN), according to the number of swollen sperm heads and pronuclei in the cytoplasm.\u003c/p\u003e \u003cp\u003eCleavage and blastocyst rates were checked at 48 hpi and 6, 7, and 8 dpi, respectively. All expanded blastocysts were fixed in 0.5% glutaraldehyde (v/v) and stored at 4\u0026deg;C for TUNEL analysis and cell-number evaluation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of Nuclear Maturation Stage\u003c/h3\u003e\n\u003cp\u003eUpon maturation, oocytes were washed in PBS-PVA, denuded from cumulus cells by gentle pipetting, fixed in 0.5% glutaraldehyde (v/v) for 15 min and stored at 4\u0026deg;C. The day of the analysis, oocytes were placed in a glass slide with 1 \u0026micro;L drop of Slowfade\u0026trade; and 5 \u0026micro;g/mL Hoechst 33342 under a coverslip. After 20 min at room temperature, chromatin configurations were analyzed at 20X augmentation by fluorescence microscopy (Eclipse 80i, Nikon Instruments Europe, Amsterdam, The Netherlands). Oocytes showing a germinal vesicle (GV) chromatin configuration were considered immature, and those showing a metaphase plate and a polar body were categorized as matured metaphase II (MII) oocytes.\u003c/p\u003e\n\u003ch3\u003eViability, Early Apoptosis, and Mortality Assessment\u003c/h3\u003e\n\u003cp\u003eFollowing the manufacturers' protocol, early apoptosis was assessed using Annexin V staining (Invitrogen\u0026copy;, Thermo Fisher Scientific, Barcelona, Spain). Denuded oocytes were placed in 100 \u0026micro;L Annexin V binding buffer droplets containing 5 \u0026micro;L of Annexin V/FITC and 1 \u0026micro;L of propidium iodide (PI; 100 \u0026micro;g/mL) and incubated at 37\u0026deg;C on a heated plate in the dark for 15 min. After washing three times in PBS-PVA, oocytes were mounted on slides in a 1 \u0026micro;L drop of Slowfade\u0026trade; and 5 \u0026micro;g/mL Hoechst 33342. Oocyte nuclei were observed at 20X augmentation with an epifluorescence microscope (Eclipse 80i, Nikon Instruments Europe, Amsterdam, The Netherlands). Oocyte status was classified into the following categories: viable (Annexin V-/PI-), early apoptotic (Annexin V+/PI-), and dead (Annexin V-/PI\u0026thinsp;+\u0026thinsp;and Annexin V+/PI+). Representative images of different categories are shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Reactive Oxygen Species (ROS) and Reduced Glutathione (GSH)\u003c/h2\u003e \u003cp\u003eMature oocytes were incubated in 10 \u0026micro;M CM-H\u003csub\u003e2\u003c/sub\u003eDCFDA (Thermo Fisher Scientific, Barcelona, Spain) and 10 \u0026micro;M Cell Tracker Blue (Thermo Fisher Scientific, Barcelona, Spain) for 30 min at 37\u0026deg;C in the dark to detect intracellular reactive oxygen species (ROS) and reduced glutathione (GSH), respectively. The oocytes were then washed thrice in PBS-PVA and placed in slides for evaluation. Fluorescence intensity was observed at 20X augmentation using epifluorescence microscopy (Eclipse 80i, Nikon Instruments Europe, Amsterdam, The Netherlands) and the signal was quantified using ImageJ 1.45s software (National Institutes of Health, Bethesda, USA).\u003c/p\u003e \u003cp\u003eRepresentative images of ROS and GSH levels in sheep oocytes are shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Membrane Potential Analysis\u003c/h2\u003e \u003cp\u003eMembrane potential was determined by incubating ocytes for 30 min at 37\u0026deg;C in 0.5 \u0026micro;M of JC-1 dye (Thermo Fisher Scientific, Barcelona, Spain). After incubation, oocytes were washed twice for 5 min and then placed on glass slides. Oocytes were examined by 20X augmentation by fluorescence microscopy (Eclipse 80i, Nikon Instruments Europe, Amsterdam, Netherlands). Relative mitochondrial membrane potential was determined as the ratio of J-aggregate to J-monomer staining intensity with ImageJ 1.45s software (National Institutes of Health, Bethesda, USA).\u003c/p\u003e \u003cp\u003eRepresentative images of JC-1 stained mitochondria in sheep oocytes is shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of Mitochondrial Distribution\u003c/h2\u003e \u003cp\u003eTo determine mitochondrial distribution patterns, oocytes were subjected to double staining with MitoTracker\u0026copy; Red CMXRos (Thermo Fisher, Barcelona, Spain), a mitochondrial-specific probe and Hoechst 33342 to stain the chromosomes. Following IVM, oocytes were incubated for 20 min in PBS-PVA plus 100 nM MitoTracker\u0026copy; Red CMXRos at 37\u0026deg;C in the dark. The oocytes were washed thrice under agitation for 5 min and mounted in slides with 1 \u0026micro;L drop of Slowfade\u0026trade; and 5 \u0026micro;g/mL Hoechst 33342. Oocytes were examined under 20X augmentation by fluorescence microscopy (Eclipse 80i, Nikon Instruments Europe, Amsterdam, Netherlands). Mitochondrial distribution was classified into two categories: abnormal mitochondrial distribution in the cytoplasm and normal distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDNA Fragmentation Assay\u003c/h2\u003e \u003cp\u003eTerminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was used to detect DNA fragmentation in MII oocytes and expanded blastocysts. Samples were fixed in 4% glutaraldehyde for 15 min and permeabilized in 0.5% Triton X-100 for 1 h at room temperature. After, the In Situ Cell Death Detection kit (Merck Life Sciences, Madrid, Spain) was used to detect DNA strand breaks following the manufacturers\u0026rsquo; instructions. Briefly, oocytes and blastocysts were placed in 30 \u0026micro;L drops of TUNEL reagent with deoxyuridine 5-trisphosphate (dUTP)-conjugated isothiocyanate fluorescein and incubated for 1 h at 37\u0026deg;C. The positive control was pre-incubated with DNAse (0.2 U/\u0026micro;L) for 1 h at 37\u0026deg;C, while the negative control was incubated in the absence of deoxynucleotidyl transferase enzyme. After that, samples were washed thrice in PBS-PVA and placed in slides in a 1 \u0026micro;L drop of Slowfade\u0026trade; with 5 \u0026micro;g/mL Hoechst 33342. Samples were evaluated at 20X magnification by epifluorescence microscopy (Eclipse 80i, Nikon Instruments Europe, Amsterdam, The Netherlands). Oocytes and blastomeres with DNA damage, e.g., with a fragmented nucleus, were classified as TUNEL-positive and those without damage as TUNEL-negative (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMeiotic Spindle Configuration Assessment\u003c/h2\u003e \u003cp\u003eMature oocytes were denuded and fixed with methanol (1:1) in PBS for 20 min at room temperature and stored in PBS-PVA at 4\u0026deg; C until use. After, oocytes were incubated in a permeabilizing solution (0.5% Triton-X-100 in PBS-PVA) for 30 min at room temperature, washed twice in PBS-PVA, and then blocked with 2% FCS in PBS for 45 min. Microtubules were detected using anti-α-tubulin (1:300) at 4\u0026deg;C overnight. After rinsing twice with 2% FCS in PBS for 5 min per wash, samples were incubated for 1 h at room temperature with secondary anti-mouse fluorescein isothiocyanate (FITC)\u0026ndash;labeled secondary antibody (1:300) and Alexa Fluor 488-labelled anti-mouse IgG antibodies (Molecular Probes, Eugene, OR, USA; 1:300) at room temperature for 30 min. Oocytes were mounted on a glass slide in a drop of Slowfade with 5 \u0026micro;g/mL Hoechst 33342 to visualize chromosomes. Oocytes were examined at 20X augmentation by fluorescence microscopy (Nikon Eclipse 80i). Oocytes with a classical symmetric barrel-shaped spindle with chromosomes aligned regularly in a compact group along the equatorial plane were considered normal. In contrast, oocytes with spindles that were disorganized, clumped, dispersed, or missing (entirely or partially) with the aberration of chromatin arrangement, clumping, or dispersal from the spindle center were considered abnormal (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometry Analysis of Cumulus Cells\u003c/h2\u003e \u003cp\u003eCumulus cells were collected from mature COCs and examined using a FlowSight\u0026reg; Imaging Flow Cytometer (Amnis, Merck-Millipore, Germany) as previously described [28]. Briefly, samples were stained with 10 \u0026micro;M YO-PRO-1 and 0.5 \u0026micro;M PI to study viability, apoptosis, and mortality. Viable cells were recorded as YO-PRO-1-/PI-, while YO-PRO-1+/PI- were deemed apoptotic. Cells stained with PI were considered dead. For mitochondrial activity, cells were incubated with 200 mM of MitoTracker\u0026trade; Deep Red (Thermo Fisher Scientific, Barcelona, Spain) for 20 min at 38.5\u0026deg;C in the dark and then stained with 10 \u0026micro;M YO-PRO-1 and 0.5 \u0026micro;M PI. Viable cells with active mitochondria were considered as MitoTracker+/YO-PRO-1-. To study ROS and GSH intracellular levels in viable cells, samples were incubated with 10 \u0026micro;M of Cell Tracker\u0026trade; Blue (Thermo Fisher Scientific, Barcelona, Spain) and 10 \u0026micro;M of CM-H\u003csub\u003e2\u003c/sub\u003eDCFDA (Thermo Fisher Scientific, Barcelona, Spain) for 30 min at 38.5\u0026deg;C followed by 0.5 \u0026micro;M PI staining. A compensation overlap was performed before each experiment, and 1000 events were acquired per sample. The raw data were analyzed using IDEAS\u0026reg; software (AMNIS), and out-of-focus cells, debris, and cell clumps were excluded from the analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using the IBM SPSS 24.0 (IBM Corp.; Armonk, NY, USA) software. Data were tested for normal distribution (Kolmogorov\u0026ndash;Smirnov, and Shapiro\u0026ndash;Wilk tests) and homogeneity of variances (Levene test). First, maximum temperature, maximum relative humidity and THI values were analyzed by factorial ANOVA followed by Bonferroni \u003cem\u003epost hoc\u003c/em\u003e test considering type of season (winter, spring, summer, and autumn) as the fixed effect. In experiment 1, oocyte viability, early apoptosis, mortality, oxidative status, mitochondrial membrane potential and distribution, DNA fragmentation, maturation, fertilization, and embryo development rates, blastocyst quality and cumulus cells activity were analyzed by factorial ANOVA followed by Bonferroni \u003cem\u003epost hoc\u003c/em\u003e test. For that, type of season was considered the fixed effect. In experiment 2, oocyte viability, early apoptosis, mortality, oxidative status, mitochondrial membrane potential and distribution, DNA fragmentation; maturation rates, meiotic spindle and chromosome organization; fertilization rates and embryo production and cumulus cells parameters were also analyzed by factorial ANOVA followed by Bonferroni \u003cem\u003epost hoc\u003c/em\u003e test. For that, ovary storage temperature (30\u0026deg;C, 38.5\u0026deg;C, 40\u0026deg;C and 41\u0026deg;C) and the replicate were considered fixed effects. Differences with probabilities of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered significant, and results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eTemperature Humidity Indices as Indicators to Heat Stress of Climatic Conditions\u003c/h2\u003e\n \u003cp\u003eThe range in maximum temperature, maximum relative humidity and THI values for each season between 2019 and 2020 is illustrated in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. THI was significantly greater (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in summer compared to winter and spring, although there was no difference with autumn.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eEXPERIMENT 1:\u003c/h2\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003eEffect of season on oocyte and cumulus cells quality parameters\u003c/h2\u003e\n \u003cp\u003eAfter IVM, the collection of COCs during summer resulted in reduced (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) oocyte viability (29.37\u0026thinsp;\u0026plusmn;\u0026thinsp;14.07%) compared to winter and autumn (84.44\u0026thinsp;\u0026plusmn;\u0026thinsp;9.38 and 84.58\u0026thinsp;\u0026plusmn;\u0026thinsp;11.49%, respectively), and a higher number (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of apoptotic oocytes (67.50\u0026thinsp;\u0026plusmn;\u0026thinsp;8.14%) in comparison to the rest of seasons (winter\u0026thinsp;=\u0026thinsp;10.00\u0026thinsp;\u0026plusmn;\u0026thinsp;7.18%; spring\u0026thinsp;=\u0026thinsp;16.67\u0026thinsp;\u0026plusmn;\u0026thinsp;7.18%; and autumn\u0026thinsp;=\u0026thinsp;11.67\u0026thinsp;\u0026plusmn;\u0026thinsp;8.79%; Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA). The percentage of dead oocytes was similar between the different seasons (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eTo determine the effect of season on ovine oocyte oxidative status, intracellular ROS and GSH were determined in the corresponding post-IVM oocytes. The levels of ROS were significantly higher (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the oocytes collected during autumn (145.97\u0026thinsp;\u0026plusmn;\u0026thinsp;20.44) than in the spring (30.38\u0026thinsp;\u0026plusmn;\u0026thinsp;16.69). Additionally, GSH was significantly greater (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in autumn (142.76\u0026thinsp;\u0026plusmn;\u0026thinsp;15.45) compared to the rest of seasons (winter\u0026thinsp;=\u0026thinsp;85.17\u0026thinsp;\u0026plusmn;\u0026thinsp;12.62; spring\u0026thinsp;=\u0026thinsp;51.55\u0026thinsp;\u0026plusmn;\u0026thinsp;12.62; and summer\u0026thinsp;=\u0026thinsp;70.01\u0026thinsp;\u0026plusmn;\u0026thinsp;14.31; Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eBoth the normal distribution of mitochondria throughout the oocyte and mitochondrial membrane potential was significantly lower (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in summer (67.14\u0026thinsp;\u0026plusmn;\u0026thinsp;7.36% and 0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, respectively) compared to winter (96.67\u0026thinsp;\u0026plusmn;\u0026thinsp;6.49%) in the former and to autumn (0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04) in the latter (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD), although there were no differences with the other seasons.\u003c/p\u003e\n \u003cp\u003eThe number of oocytes with fragmented DNA was higher during summer (25.92\u0026thinsp;\u0026plusmn;\u0026thinsp;8.15%) than the rest of seasons (winter\u0026thinsp;=\u0026thinsp;11.42\u0026thinsp;\u0026plusmn;\u0026thinsp;7.19%; spring\u0026thinsp;=\u0026thinsp;1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;7.19%; and autumn\u0026thinsp;=\u0026thinsp;0\u0026thinsp;\u0026plusmn;\u0026thinsp;8.80%) although the difference was not statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003eAs shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, cumulus cells live/death status was significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduced during summer compared to spring since a lower number of viable cells and a higher number of dead cells were found between these two seasons, although differences were no found between summer and winter and autumn. We also assessed cumulus cells\u0026apos; apoptosis, number of active mitochondria, and intracellular ROS and GSH content. In all parameters, oocytes showed similar values (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) throughout the whole year (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of season on subsequent maturation and developmental competence of sheep oocytes\u003c/h2\u003e\n \u003cp\u003eResults did not show significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in the maturation and fertilization rates (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Although the oocytes collected throughout the four seasons had similar cleavage rates (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), the percentage of blastocysts from the initial number of oocytes was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in winter compared to summer and autumn (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In addition, the number of blastocysts from the cleaved embryos at 48 hpi was also significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) during winter and spring compared to summer and autumn (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eIn vitro blastocysts produced during summer show reduced quality\u003c/h2\u003e\n \u003cp\u003eAlthough the number of cells of \u003cem\u003ein vitro\u003c/em\u003e produced blastocysts was similar (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) among all seasons (winter\u0026thinsp;=\u0026thinsp;122.02\u0026thinsp;\u0026plusmn;\u0026thinsp;8.69%; spring\u0026thinsp;=\u0026thinsp;127.11\u0026thinsp;\u0026plusmn;\u0026thinsp;7.34%; summer\u0026thinsp;=\u0026thinsp;117.61\u0026thinsp;\u0026plusmn;\u0026thinsp;7.93%; and autumn\u0026thinsp;=\u0026thinsp;104.11\u0026thinsp;\u0026plusmn;\u0026thinsp;8.69%), the percentage of blastomeres showing DNA fragmentation was higher during summer (19.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.71%) compared to winter (6.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97%) and spring (8.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.51%; Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e) although there was no difference with autumn.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eEXPERIMENT 2:\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003eIncreasing the ovary storage temperature in an in vitro ovary storage model impairs the quality of sheep oocytes and cumulus cells\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eA, the results revealed that the storage of slaughterhouse sheep ovaries for 3 h at 30\u0026deg;C produced greater (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) oocyte viability values (73.44\u0026thinsp;\u0026plusmn;\u0026thinsp;6.48%) in comparison with 40\u0026deg;C (34.23\u0026thinsp;\u0026plusmn;\u0026thinsp;8.29%) and 41\u0026deg;C (16.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.29%; Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eA). Additionally, the lowest (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) percentages of dead oocytes were observed at 30\u0026deg;C (9.37\u0026thinsp;\u0026plusmn;\u0026thinsp;5.84%) and 38.5\u0026deg;C (29.68\u0026thinsp;\u0026plusmn;\u0026thinsp;5.84%) in contrast to 41\u0026deg;C (69.55\u0026thinsp;\u0026plusmn;\u0026thinsp;8.05%; Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eIntracellular ROS levels were higher at 30\u0026deg;C (91.20\u0026thinsp;\u0026plusmn;\u0026thinsp;10.99) although there was no difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) with 38.5\u0026deg;C, 40\u0026deg;C and 41\u0026deg;C (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eB). Moreover, the values for the GSH content were similar between temperatures (30\u0026deg;C\u0026thinsp;=\u0026thinsp;95.64\u0026thinsp;\u0026plusmn;\u0026thinsp;12.41; 38.5\u0026deg;C\u0026thinsp;=\u0026thinsp;65.33\u0026thinsp;\u0026plusmn;\u0026thinsp;12.41; 40\u0026deg;C\u0026thinsp;=\u0026thinsp;44.68\u0026thinsp;\u0026plusmn;\u0026thinsp;17.10 and 41\u0026deg;C\u0026thinsp;=\u0026thinsp;86.57\u0026thinsp;\u0026plusmn;\u0026thinsp;17.10; Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eRegarding the mitochondria, although the membrane potential in terms of fluorescence intensity did not show significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) among temperatures (30\u0026deg;C\u0026thinsp;=\u0026thinsp;0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02; 38.5\u0026deg;C\u0026thinsp;=\u0026thinsp;0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02; 40\u0026deg;C\u0026thinsp;=\u0026thinsp;0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 and 41\u0026deg;C\u0026thinsp;=\u0026thinsp;0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03), the normal distribution of these organelles was higher at 30\u0026deg;C and 38.5\u0026deg;C (87.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.43% and 82.68\u0026thinsp;\u0026plusmn;\u0026thinsp;6.43%, respectively) than at 41\u0026deg;C (39.02\u0026thinsp;\u0026plusmn;\u0026thinsp;8.87%; Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eThe number of oocytes with fragmented DNA as measured by the TUNEL assay was significantly lower (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after an ovary storage at 30\u0026deg;C (5.21\u0026thinsp;\u0026plusmn;\u0026thinsp;7.83%) when compared to the rest of temperatures (38.5\u0026deg;C\u0026thinsp;=\u0026thinsp;56.06\u0026thinsp;\u0026plusmn;\u0026thinsp;7.83%; 40\u0026deg;C\u0026thinsp;=\u0026thinsp;55.44\u0026thinsp;\u0026plusmn;\u0026thinsp;10.79% and 41\u0026deg;C\u0026thinsp;=\u0026thinsp;60.84\u0026thinsp;\u0026plusmn;\u0026thinsp;10.79%; Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eD).\u003c/p\u003e\n \u003cp\u003eAs shown in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, the percentage of viable cumulus cells was significantly higher (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at 30\u0026deg;C compared to 38.5\u0026deg;C and 40\u0026deg;C, although there was no different in relation to 41\u0026deg;C. The number of active mitochondria in cumulus cells was significantly greater (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) at 30\u0026deg;C compared to 38.5\u0026deg;C, 40\u0026deg;C and 41\u0026deg;C. However, the rest of parameters (intracellular ROS and GSH content) showed similar values (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between temperatures.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003eStage of the nuclear maturation, meiotic spindle, and chromosome organization with increasing ovary storage temperature\u003c/h2\u003e\n \u003cp\u003eThe influence of temperature on oocyte nuclear maturation, cytoskeletal integrity, and chromosome organization after IVM was assesed. The rate of oocytes reaching MII phase was greater (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the 30\u0026deg;C group (69.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.97%) compared to the rest of temperatures (38.5\u0026deg;C\u0026thinsp;=\u0026thinsp;21.73\u0026thinsp;\u0026plusmn;\u0026thinsp;4.67%; 40\u0026deg;C\u0026thinsp;=\u0026thinsp;16.87\u0026thinsp;\u0026plusmn;\u0026thinsp;6.43%; and 41\u0026deg;C\u0026thinsp;=\u0026thinsp;9.82\u0026thinsp;\u0026plusmn;\u0026thinsp;6.43%; Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eA). Given the significantly compromised maturation rates at 40\u0026deg;C and 41\u0026deg;C, the evaluation of meiotic spindle configuration was limited to the 30\u0026deg;C and 38.5\u0026deg;C groups. This limitation was necessary, as the low maturation rates at elevated temperatures made the reliable assessment of spindle integrity unfeasible. As expected, most of the 30\u0026deg;C group oocytes contained a typical MII spindle (91.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.09%; Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eB). However, in the 38.5\u0026deg;C group a reduced (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) number of oocytes (49.41\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79%) presented a normal meiotic spindle configuration. Regardless, the proportion of oocytes with a normal organization of chromosomes was not statistically different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between the temperature\u0026rsquo;s groups (30\u0026deg;C\u0026thinsp;=\u0026thinsp;90.96\u0026thinsp;\u0026plusmn;\u0026thinsp;5.61%; and 38.5\u0026deg;C\u0026thinsp;=\u0026thinsp;67.53\u0026thinsp;\u0026plusmn;\u0026thinsp;6.88%; Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eB).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003eIncreasing ovary storage temperature reduces in vitro embryo production rates and blastocyst quality in sheep\u003c/h2\u003e\n \u003cp\u003eWhile no differences were observed in the fertilization rates (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), the results reported in Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e show that the oocytes collected from ovaries stored at 30\u0026deg;C yield cleavage rates higher (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than at 38.5\u0026deg;C (only these 2 temperatures were studied because of the extremely low number of matured oocytes for 40\u0026deg;C and 41\u0026deg;C). Moreover, the number of expanded blastocysts from the total of oocytes in culture and the cleaved embryos at 48 hpi followed the same tendency and were significantly higher at 30\u0026deg;C compared with 38.5\u0026deg;C (Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Although the blastocyst total cell number was similar (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) among temperatures, the use of 38.5\u0026deg;C increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the percentage of blastomere DNA fragmentation compared to 30\u0026deg;C.\u003c/p\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe ever-growing global population is driving a notable increase in global food production \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Annual meat production, and particularly sheep meat demand in Europe, is expected to increase due to dietary diversification and demographic changes \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, highlighting the growing importance of sheep and goat farming in the future \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. At a time when increasing livestock production and productivity is key to meeting the escalating demand for animal protein globally, the climate is changing faster than the predictions, posing a significant challenge to the long-term viability of livestock production systems \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMammals are able to maintain body temperatures higher than environmental (35\u0026ndash;39\u0026deg;C) through a balance of body heat production and loss \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and global warming can affect the proper functioning of that metabolism. Changes in ambient temperature over recent years and especially the increase in the global average surface temperature of about 0.6\u0026deg;C over the past 20th century can upset the balance \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Temperature has a clear effect on mammalian gamete's function, which shows high sensitivity to heat stress \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e in both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e systems \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Extreme high-temperature conditions can increase body temperature and adversely impact mammalian biological functions, leading to impaired production and reproductive traits \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Most reproductive processes, including gametogenesis, fertilization or embryonic development, can be influenced by extreme high environmental temperatures that directly impact reproductive performance \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Even though sheep are found to be well adapted to different environmental conditions including high environmental temperatures \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, heat stress significantly impairs reproduction and entails a risk to the efficiency of meat production \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo meet increasing global demands and minimize environmental consequences, advances in animal agriculture via reproductive technologies will be essential. These technologies aim to enhance livestock efficiency and productivity while adjusting to climate change and global warming \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Nowadays, assisted reproduction techniques are widely used in livestock, though IVP has been little explored because of its poor performance \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe collection of developmentally competent oocytes is a drawback that may limit IVP applicability. Several studies have demonstrated that heat stress adversely impacts on fertility process, especially in oogenesis, oocyte function, maturation, fertilization, and blastocyst development \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The molecular study of oocyte damage triggered by heat stress could help to avoid this type of injury. The present work compares, for the first time, whether the effects of heat stress season-dependent (winter, spring, summer, and autumn) are replicated in the \u003cem\u003ein vitro\u003c/em\u003e model which is crucial for a deeper understanding of the physiological mechanisms underlying oocyte damage. Thus, we evaluate the evolution of oocyte competence, fertility and blastocyst rates throughout the seasons and the effects of high \u003cem\u003ein vitro\u003c/em\u003e temperatures on oocyte quality and its competence for \u003cem\u003ein vitro\u003c/em\u003e embryo production.\u003c/p\u003e \u003cp\u003eDifferent authors have shown that the quality of the oocyte, and consequently the production of embryos, is affected by seasonality in small ruminants. Souza-Fabjan et al., \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e showed that blastocyst production in goats was higher during fall and lower in spring. On the other hand, Serra et al., \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e demonstrated a seasonal effect in sheep with better oocyte quality results in spring compared to autumn, although this study was carried out in prepubertal animals where the endocrine profile is quite different from adults \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In our case, the data generally showed a poorer oocyte quality for the summer months. We hypothesize that this fact was due to the high temperatures registered in our country at that time of the year. In this way, Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows that the level of heat stress reached during summer was considered to be severe (79\u0026ndash;83) to ruminant species, while the rest of seasons showed values considered as normal (\u0026le;\u0026thinsp;74). We think that the low quality shown during summer is not due to a photoperiod seasonal effect since some parameters analyzed showed better results during spring which is a non-optimal reproductive season for sheep species in our latitude. Therefore, what should have been expected during spring considering the photoperiod is a worse quality also at this time of the year.\u003c/p\u003e \u003cp\u003eApoptosis is a form of programmed cell death that plays a crucial role in maintaining the homeostasis of various biological processes \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e and plays an important role in the disruption of the normal function of oocytes under thermal stress \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In this study, Annexin-V staining has been used as an early marker of apoptosis, detecting alterations in the oocyte phospholipid membrane and preceding late stages of apoptosis assessed by TUNEL staining. The early apoptosis, assessed using the V-FITC assay, is more frequent in oocytes subjected to heat stress, suggesting that summer high temperatures can induce apoptosis in oocytes \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In fact, our results reported by Annexin-V staining showed that the percentage of viable oocytes decreases during summer, while the apoptotic oocyte rate increases. Likewise, Ahmadi \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e observed a significant interaction between season and thermal stress on apoptosis in sheep, where the reduced developmental competence observed in heat-stressed oocytes was partially linked to changes in their plasma membrane. Similarly, the percentage of viable oocytes decreased for higher \u003cem\u003ein vitro\u003c/em\u003e temperatures (40\u0026deg;C and 41\u0026deg;C) in relation to 30\u0026deg;C. Our results are in agreement with previous studies performed in bovine where oocytes subjected to heat stress before 12 h maturation \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, or even in the short-term at the early stage of maturation, resulted in increased Annexin-V binding and oocytes that undergo early apoptosis compared to the control group \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHeat stress has been shown to activate the apoptotic cascades, inducing alterations in the oocyte phospholipid membrane \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e and ultimately promoting a detrimental on oocyte developmental competence \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. However, in our results the stress caused by the environmental temperature was not enough to damage oocyte DNA, although \u003cem\u003ein vitro\u003c/em\u003e temperatures over 38.5\u0026deg;C dramatically damaged the oocyte DNA. The different effect observed between ambient heat stress and \u003cem\u003ein vitro\u003c/em\u003e heat stress may be caused by the differences in the response of both models to high temperatures. Under physiological systems, provided the stress is not excessive, the organism's defense system will deal with potential injuries \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Thus, during the summer months, early apoptosis greatly increases unlike dead cell percentage or DNA damage. Early apoptosis is an event that can be reversed by the body's defense systems as the synthesis of antiapoptotic proteins or the synthesis of antioxidants \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Indeed, some studies have shown that heat shock induces early apoptosis events though upstream of DNA fragmentation \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. This \u003cem\u003ein vivo\u003c/em\u003e adaptational phenomenon was previously noted in bovine \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e and sheep \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e oocytes and even spermatozoa \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. The development of heat tolerance could be associated with the expression of heat shock proteins, such as heat shock protein-70, which protects oocytes from apoptotic stimuli that harmfully affect DNA \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Thus, it would also be of interest to develop future experiments to determine if heat stress significantly impacts the expression levels of specific genes for both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models. Despite the result observed during the seasons, after excessive \u003cem\u003ein vitro\u003c/em\u003e heat stress, the oocyte may not be able to counteract this damage as it may not have an active defense system, accumulating irreversible damage that leads to higher dead cell percentage and DNA damage. Actually, our findings are in line with other works where ovaries exposed to severe \u003cem\u003ein vitro\u003c/em\u003e heat stress (high storage temperature and/or long-term storage) increased the number of oocytes with DNA fragmented nuclei \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRegarding the oxidative balance, our results have shown that GSH and ROS production was higher during autumn compared to the rest of the seasons for the former parameter and compared to spring for the latter as previously reported \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The balance between ROS production and antioxidant capacity also affects the developmental competence of oocytes. Under physiological conditions, ROS regulate specific cellular functions while high levels lead to various forms of cellular damage to DNA, proteins, lipids and ultimately affecting oocyte quality and viability \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Although an excess of ROS may lead to a harmful effect on oocytes, it has been shown that the presence of ROS derived from mitochondrial respiration is necessary for certain cell signaling pathways involved in folliculogenesis, oocyte maturation, embryogenesis and implantation \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. In our latitude, the reproductive season in small ruminants begins from the end of summer to the beginning of winter. This fact drives us to think that during this period, higher cellular activity will be needed, the mitochondria will be more active, and more ROS will be generated. Additionally, our results are in line with other studies performed on sheep under similar latitudes, where oocytes collected during autumn also showed a significantly higher ROS production compared with other seasons \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Among the main antioxidants that protect the oocyte against oxidative damage, GSH plays a key role, showing the cytoplasmic maturity degree and the quality of the oocyte after \u003cem\u003ein vitro\u003c/em\u003e maturation \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. The regulation of intracellular redox potential in the oocyte is a crucial determinant of fertility and embryo development \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Thus, the high concentrations of GSH observed during autumn, compared with the rest of the seasons, could have been increased in order to achieve an oxidative balance and physiologically counteract the high production of ROS. Similarly, other works have also reported that animals may respond to temperature challenges by upregulating antioxidant enzymes \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Moreover, variation in antioxidant levels was suggested not only as a defense mechanism in order to counteract oxidative damage but also as a consequence of environmental conditions \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Regarding the effect of \u003cem\u003ein vitro\u003c/em\u003e heat stress, the temperature increase did not affect the production of ROS and GSH. This may be due to the high percentage of dead oocytes that we found for temperatures over 38.5\u0026deg;C where the physiological system that determines the oxidation state of a cell could not be activated. Furthermore, we hypothesize that it is possible that live oocytes were unable to display the mechanisms that lead to the production of antioxidant enzymes due to the damage produced, hence this effect could not be visualized \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConcerning mitochondrial parameters, it is well known that a dysfunction in one or more aspects of mitochondrial biology results in reduced oocyte developmental competence. We observed that both mitochondrial activity and the homogeneous distribution of mitochondria were affected throughout the seasons. Moreover, this study shows not only heat stress-induced alterations in mitochondrial distribution \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e but also observes a trend where homogeneous mitochondrial distribution aligns with mitochondrial membrane potential during summer. Thus, mitochondrial activity was higher during the autumn season compared to spring and summer and the percentage of oocytes with a normal distribution of mitochondria was lower in summer than in winter. These results agree with other studies where mitochondrial distribution differs between cold and warm seasons \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Particularly, our findings are aligned with previous research indicating that mitochondria exhibit less uniform distribution during summer \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e or afterward severe \u003cem\u003ein vitro\u003c/em\u003e heat exposure (41\u0026deg;C) \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This fact could be due to an alteration of the cytoskeletal proteins caused by heat, as has been previously demonstrated by Gendelman and Roth (2012). Extremely high temperatures have been shown to denature cytoskeletal proteins that leads to its abnormal distribution in the oocyte \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e, leading mitochondria to relocate to the cell periphery instead of maintaining a homogeneous distribution throughout the cytoplasm \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. This alteration of the cytoskeletal elements can even lead to the disruption of nuclear maturation and the disassembly of the meiotic spindle \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is well known that meiotic spindle microtubules are susceptible to temperature changes which can ultimately cause chromosomal imbalance and cell death \u003csup\u003e\u003cspan additionalcitationids=\"CR74\" citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. The microtubular network in the oocyte is critical for meiotic spindle formation, chromosome segregation, fertilization, and embryonic development \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Microtubules and their post-translational modification, especially tyrosination and acetylation in sheep oocytes \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e, play a crucial role in oocyte maturation and fertilization due to their influence on meiotic spindle assembly and chromosome movement. A balance between tyrosination (more abundant in dynamic microtubules) and acetylation (characterizing stability) is essential for the normal function of the meiotic spindle, which requires both dynamism and stability. Nevertheless, temperature has been shown to disrupt this balance, potentially affecting oocyte developmental competence \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Several studies have demonstrated that \u003cem\u003ein vitro\u003c/em\u003e heat stress has detrimental effects on various aspects of cytoplasmic and nuclear oocyte maturation \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e, including cytoskeletal rearrangement \u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e, meiotic spindle formation \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e and early embryonic mitotic failures \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. In the present study, we also observed that the meiotic spindle was affected by a short exposure for 3 hours at 38.5\u0026deg;C, resulting in a lower percentage of oocytes with a normal spindle configuration. This parameter had a clear association with the low maturation rates of high temperatures (from 38.5\u0026deg;C). However, oocyte maturation was not affected by the temperatures recorded throughout the seasons. Unfortunately, the spindle configuration could not be assessed in this experiment therefore no data are available on this parameter. Our findings are consistent with other studies conducted on bovine oocytes which reported no significant seasonal effects on nuclear maturation \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e and a normal uniform alignment of the chromosomes on the spindle \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRegarding the cleavage rate, there were no differences between seasons, although the blastocysts percentage was lower during the summer and autumn. The fact that oocytes obtained in the summer season showed no differences with those collected during the rest of the year in terms of nuclear maturation and cleavage rate could reflect that immature oocytes can be fertilized and undergo cleavage without developing to the blastocyst stage \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In addition, the embryos obtained during those months had a higher number of apoptotic cells compared to winter and autumn. Similar results were found by Gendelman and Roth (2012). Furthermore, genetic studies suggest that heat stress during the summer months impedes embryo development, likely due to altered expressions in these conditions associated with a reduced developmental capacity of oocytes and embryos \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. On the other hand, storing the ovaries for 3 hours at temperatures above 38.5\u0026deg;C produced a lower percentage of cleavage and blastocysts compared to 30\u0026deg;C in alignment with previous studies \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This decrease is likely to be a consequence of hyperthermia. It is important to note that these blastocysts were obtained from oocytes subjected to heat stress, which was previously observed to affect their quality and developmental competence in sheep \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The nuclear maturation and spindle configuration were significantly impaired for oocytes exposed to \u003cem\u003ein vitro\u003c/em\u003e heat stress compared to their unexposed counterparts. This impairment could potentially result in irreparable damage to the percentage of oocytes progressing past the cleavage stage, leading to a subsequent decline in embryo developmental potential \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Similarly, the number of apoptotic cells was higher for blastocysts from ovaries stored at 38.5\u0026deg;C. These results agree with those obtained by Gendelman and Roth (2012a, b) \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e,\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCumulus cells play critical roles in the maturation of the nucleus and cytoplasm in the oocyte since they allow the interchange of molecules with the oocytes \u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. In addition, they support energy production in the COCs and protect against oxidative stress-induced apoptosis. It was demonstrated that CCs are also important for the thermal protection of oocytes, as they provide extracellular thermoprotective molecules through gap junctions \u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e,\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. In addition, CCs also produce regulatory molecules that activate thermoprotective mechanisms within the oocyte \u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. Nevertheless, heat stress has been reported to reduce the effectiveness of the gap junction communication between cumulus cells and oocytes \u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. As expected, we have reported a lower percentage of viable CCs during the summer season as well as a higher percentage of dead cells compared with other seasons. Nevertheless, apoptotic percentage, ROS/GSH levels and mitochondrial activity were not affected. On the other hand, a lower viable percentage for the temperature 38.5\u0026deg;C was observed in support of oocyte apoptosis assay results. In this line, different studies have seen that the apoptosis incidence in cumulus cells is negatively correlated to the developmental competence of oocytes and subsequent embryonic development after fertilization \u003csup\u003e\u003cspan additionalcitationids=\"CR88\" citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e. Nevertheless, other works showed that oocytes were more sensitive to heat stress than cumulus cells \u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e and just a long exposure to heat stress conditions may overwhelm the cumulus cells protection capacity \u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e. Moreover, although we have not studied morphological defects, cumulus morphology can also be affected by \u003cem\u003ein vitro\u003c/em\u003e heat stress during ovaries storage \u003csup\u003e\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e as well as during summer rather than in winter \u003csup\u003e\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, no studies have simultaneously evaluated the effects of collection season on oocyte quality and IVP in sheep while also monitoring the impact of varying ovarian storage temperatures on oocyte quality and competence. The results revealed that extreme environmental temperatures during summer significantly decreased oocyte and cumulus cells viability, DNA integrity and mitochondrial normal distribution. Moreover, ovary collections during summer had not only lower blastocyst percentage but also summer-produced blastocysts exhibited significantly higher DNA fragmentation compared to winter. This harmful effect was not limited to summer seasons since it was carried over to autumn, indicating a carry-over effect caused by heat stress during the summer. Similarly, the severity of the damages caused during \u003cem\u003ein vitro\u003c/em\u003e heat stress was related to the highest temperatures selected, where temperatures over 38.5\u0026deg;C caused major injuries on oocyte quality parameters assessed and blastocyst rates. These findings clearly demonstrated that both seasonal and \u003cem\u003ein vitro\u003c/em\u003e extreme temperatures significantly impaired oocyte quality and embryo production, emphasizing the need to develop strategies to reduce the impacts of heat stress and enhance reproductive outcomes in sheep.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: AJS.; methodology: AM-M, IS-A, MI-C, D-AM-C, CM and RF-S; formal analysis: AM-M and IS-A; resources: AJS and JJG.; writing: AM-M, IS-A and AJS; original draft preparation: AM-M; writing\u0026mdash;review and editing: AJS and JJG; funding acquisition: AJS and JJG. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Spanish Ministry of Economy and Competitiveness (AGL2017-89017-R). AM-M. was supported by a Ministry of Economy and Competitiveness scholarship (PRE2018-084837). D-AM-C received a contract from UCLM cofinanced by the European Social Fund.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe adult sheep ovaries were collected from an authorized slaughterhouse, and sperm samples were obtained from the Germplasm Bank of the \u0026ldquo;Reproduction Biology Group\u0026rdquo;, which is officially authorized for collecting and storing semen from sheep (ES07RS02OC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlexandratos, N. \u0026amp; Bruinsma, J. 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Minireview: Interactions between somatic cells and germ cells throughout mammalian oogenesis. \u003cem\u003eBiol Reprod\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 543\u0026ndash;547 (1990).\u003c/li\u003e\n\u003cli\u003eEdwards, J. L. \u0026amp; Hansen, P. J. Elevated temperature increases heat shock protein 70 synthesis in bovine two-cell embryos and compromises function of maturing oocytes. \u003cem\u003eBiol Reprod\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 340\u0026ndash;346 (1996).\u003c/li\u003e\n\u003cli\u003eCampen, K. A. \u003cem\u003eet al.\u003c/em\u003e Heat stress impairs gap junction communication and cumulus function of Bovine Oocytes. \u003cem\u003eTheriogenology\u003c/em\u003e \u003cstrong\u003e64\u003c/strong\u003e, 2\u0026ndash;9 (2018).\u003c/li\u003e\n\u003cli\u003eYuan, Y. 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M. \u003cem\u003eet al.\u003c/em\u003e Thermotolerance and plasticity of camel somatic cells exposed to acute and chronic heat stress. \u003cem\u003eJ Adv Res\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 105\u0026ndash;118 (2020).\u003c/li\u003e\n\u003cli\u003eP\u0026ouml;hland, R. \u003cem\u003eet al.\u003c/em\u003e Influence of long-term thermal stress on the in vitro maturation on embryo development and Heat Shock Protein abundance in zebu cattle. \u003cem\u003eAnim Reprod\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1\u0026ndash;10 (2020).\u003c/li\u003e\n\u003cli\u003ePedersen, H. G., Watson, E. D. \u0026amp; Telfer, E. E. Effect of ovary holding temperature and time on equine granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology. \u003cem\u003eTheriogenology\u003c/em\u003e \u003cstrong\u003e62\u003c/strong\u003e, 468\u0026ndash;480 (2004).\u003c/li\u003e\n\u003cli\u003eAhmadi, E., Nazari, H. \u0026amp; Hossini-Fahraji, H. Low developmental competence and high tolerance to thermal stress of ovine oocytes in the warm compared with the cold season. \u003cem\u003eTrop Anim Health Prod\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, 1611\u0026ndash;1618 (2019).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ovine, high temperature, oocyte quality, embryo quality, temperature humidity index","lastPublishedDoi":"10.21203/rs.3.rs-5595754/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5595754/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlobal population growth requires an increase in food production, particularly meat, with an expected rise in sheep farming. However, climate change challenges livestock management, with heat stress negatively impacting reproductive performance. \u003cem\u003eIn vitro\u003c/em\u003e embryo production (IVP) in sheep farming is promising, though optimizing embryo quality and efficiency remains challenging. Heat stress impairs oocyte developmental competence, affecting IVP outcomes. This study investigates the effect of season given seasonal variations in temperature and temperature humidity index (THI) and \u003cem\u003ein vitro\u003c/em\u003e induced heat stress on oocyte quality and embryo production. In the first experiment, ovaries were collected in the four seasons (winter, spring, summer and autumn) with differences in THI and in the second experiment ovaries were exposed to 30\u0026deg;C (control), 38.5\u0026deg;C, 40\u0026deg;C and 41\u0026deg;C. Results indicate that elevated summer temperatures significantly compromise oocyte and cumulus cell viability, DNA integrity, mitochondrial distribution, and blastocyst quality. These detrimental effects persisted into autumn, likely due to a carry-over effect from summer heat stress. Furthermore, \u003cem\u003ein vitro\u003c/em\u003e exposure to temperatures at or above 38.5\u0026deg;C led to marked declines in oocyte quality and blastocyst rates. Understanding these effects is essential for developing strategies to mitigate heat stress and enhance reproductive outcomes in sheep.\u003c/p\u003e","manuscriptTitle":"How Heat Stress Affects the Functionality of the Ovine Cumulus-Oocyte Complex: Implications for In Vitro Embryo Production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-20 22:47:02","doi":"10.21203/rs.3.rs-5595754/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-03T05:36:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-01T00:36:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-24T13:36:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92679468414342892632527960346846179626","date":"2025-01-11T08:48:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300237484575349741609678891356413817829","date":"2025-01-09T16:56:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-05T16:36:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28563086611102939516893091781190140794","date":"2024-12-26T20:04:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-21T15:04:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-21T14:51:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-12-20T02:02:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-18T12:09:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-12-06T19:11:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2753a1ab-258d-4def-9aa6-55de4839f090","owner":[],"postedDate":"December 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T16:01:11+00:00","versionOfRecord":{"articleIdentity":"rs-5595754","link":"https://doi.org/10.1038/s41598-025-01173-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-05-17 15:57:05","publishedOnDateReadable":"May 17th, 2025"},"versionCreatedAt":"2024-12-20 22:47:02","video":"","vorDoi":"10.1038/s41598-025-01173-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-01173-1","workflowStages":[]},"version":"v1","identity":"rs-5595754","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5595754","identity":"rs-5595754","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}