The Implementation of Disaster Management Life Cycle During the Earthquake 2005 in Kashmir Pakistan and the Disaster Response of EMS Services

Disaster Management Plan [1]

Organizations involved:
1. Earthquake Reconstruction & Rehabilitation Authority Pakistan (ERRA since 2005)
2. NDMA (National Disaster management authority Pakistan (Since 2010)
3. US Marine and Army helicopters from Afghanistan, Pakistan Army and retired.
4. UN, WHO, EU, OIC, UNDP, UNESCO, UNICEF, Oxfam, ICRC-red crescent, JEN-Japan Others.

Management Cycle

(Figures 1 & 2) [2].

Figure 1: Management cycle (Google Disaster life cycle, 2021).

Figure 2: Management cycle (Google Disaster life cycle, 2021).

Kashmir Earthquake 2005 [3]

The 2005 Kashmir earthquake occurred at 08:50 am Pakistan Standard (Wikipedia, 2021) Time on 8 October in Pakistani Azad Kashmir. It registered a moment magnitude of 7.6 and had a maximum Mercalli intensity of VIII (Severe) or XI (Extreme). The earthquake also affected countries in the surrounding region where tremors were felt in Afghanistan, Tajikistan, India and the China Xinjiang region. The severity of the damage caused by the earthquake is attributed to severe upthrust. Over 86,000 people died a similar 100,000 number were injured and millions were displaced. It is considered the deadliest earthquake to hit South Asia (Table 1).

Table 1: The magnitude of Disaster because of Earthquake 2005 in Kashmir Pakistan and disaster lifecycle.

Preparation [4]

Disaster preparedness: 5 key components to effective emergency [4] management were used in the 2005 Kashmir earthquake.
1. Clear communication.
2. Comprehensive training.
3. Knowledge of assets.
4. Technology fail-safes and protocol.
5. Healthcare leadership involvement. Disaster response in the early phase of [5] earthquake relief is complex with local facilities often overwhelmed and damaged. Coordinated effort is required for success with lessons learnt to improve future disaster management.

Financial Assistance and Aid [6]

In late 2006 a staggering $20 billion USD development scheme was mooted [6] by Pakistan for reconstruction and rehabilitation of the earthquake hit zones in Azad Kashmir. A land use plan for Muzaffarabad city had been prepared by Japan International Cooperation Agency. Countries of Asia, Africa, EU, Americas, Oceana, Multinational organizations, NGOS - On November 19, 2005, it was estimated that the international community as a whole pledged about US$5.8 billion. (Wikipedia 2005 Kashmir earthquake, 2021) [7] (Figures 3-5).

Figure 3: (Wikipedia 2005 Kashmir earthquake, 2021).

Figure 4: (Wikipedia 2005 Kashmir earthquake, 2021).

Figure 5: (Wikipedia International response, 2021).

Mitigation [8]

Risk mitigation refers to the process of planning and developing [8] methods and options to reduce threats or risks to project objectives. The following five strategies can be used in risk mitigation planning and monitoring.
1. Assume and accept risk.
2. Avoidance of risk.
3. Controlling risk.
4. Transference of risk.
5. Watch and monitor risk.
Although susceptibility zoning maps represent [9] a powerful tool in natural hazard management caution is needed when developing and using such maps. The October 2005 earthquake triggered several thousand landslides in the Lesser Himalaya of Kashmir in northern Pakistan and India. Preliminary results from repeat photographs from 2005 and 2006 after the snowmelt season reveal that much of the ongoing land sliding occurred along rivers and roads, and the extensive earthquake-induced fissuring. Although the susceptibility zoning success rate for 2001 was low many of the co post seismic land sliding in 2005 occurred in areas that had been defined as being potentially dangerous on the 2001 map [9]. Within a designated study area of 2250 km2 the number of landslides increased from 369 in 2001 to 2252 in October 2005.

Response [10]

Disaster response is the assistance and intervention [10] during or immediately after an emergency or disaster. Focus is on saving lives and protecting community assets (buildings, roads, animals, crops, infrastructure). Usually measured in hours, days or weeks. Immediately after the earthquake occurred the largest rescue and relief [11] operation was launched in the history of Pakistan. The Pakistani Army was directed to extend help to the civilian population in the quake affected areas and all civilian and military hospitals were directed to deal with the situation on an emergency basis. Many countries international organizations, and nongovernmental organizations offered relief aid to the region in the form of donations as well as relief supplies including food, medical supplies, tents, and blankets. International rescue and relief workers brought rescue equipment including helicopters and rescue dogs.

Recovery [12]

During the recovery period, restoration efforts occur concurrently with regular operations and activities. Preventing or reducing stress related illnesses and excessive financial burdens. Rebuilding damaged structures and reducing vulnerability to future disasters. It posed unique challenges and efforts on a massive scale for [13] reconstruction. For residential buildings the Pakistan government adopted a house owner driven approach. The reconstruction policy stated that the government and other agencies would provide equal technical assistance and subsidy to each family without differentiating between who lost what. To increase capacity in earthquake resistant construction large scale training of artisan’s technicians, engineers, and community mobilisers has been conducted. Campaigns to “build back better” have raised awareness in the communities. Local Housing Reconstruction Centers have been established for training advice and [13] dissemination of earthquake resistant technology. This decentralized approach has helped in achieving reconstruction smoothly [14].

Conclusion

To conclude, the importance of disaster medicine and disaster management is well recognized in last decades. The role of disaster management cycle with steps, preparation, mitigation, response and recovery with detail efforts enables the EMS of the countries to help during disaster prevention and recovery.

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Perception of Quality of Life of People with Kidney Transplantation and Transplant Candidates in Merida, Yucatan, Mexico

Introduction

Chronic kidney disease (CKD) affects around 11% of the population over the age of 20 globally, with an increase in incidence in recent years [1]. Peritoneal dialysis, hemodialysis and kidney transplantation are treatments that have been effective in increasing the life expectancy of people with CKD [1,2]. In the last three decades, the analysis of quality of life has been integrated as an indicator of the evolution of health status in patients with CKD to see beyond the number of years of survival. Quality of life is, according to the WHO, “the perception that an individual has of his place in existence, in the context of the culture and value system in which he lives and in relation to his objectives, his expectations, his norms, his concerns. It is a concept that is influenced by the physical health of the subject, their psychological state, their level of independence, their social relationships, as well as their relationship with the environment.” This concept encompasses objective and subjective aspects that reflect the degree of physical, emotional, social and economic well-being of each individual. The analysis of the quality of life in people with CKD allows us to understand the impact of the disease and its treatment, to know more about patients, how they evolve and how they adapt to organic alteration [3,4]. Currently, the analysis of quality of life in people with CKD seeks to generate evidence, qualitative and quantitative, to facilitate: the process of assessing human needs and the implementation of quality interventions in the care sectors [5]. In the health sciences, phenomenological research, and those with a qualitative approach in general, generate evidence that serves as a guide to practice sensitive to the realities of the people to whom care is directed, to their cultural diversity and to the contexts in which their lives unfold [6,7].
In studies related to quality of life in transplanted people and candidates for kidney transplantation, participants manifest as the main human responses: recurrent hospitalizations, uncertainty about the work situation, deterioration of body image, deterioration of sexual functionality, dependence on third parties, stress and guilt [2,8-12]. Specifically, people who are candidates for kidney transplantation manifest as the main human responses: anxiety and depression [13,14]. Transplanted individuals report acute rejections, medication side effects, and emotional instability; [12-15,16] immediately, after transplantation, they may perceive release with respect to dependence on renal replacement therapy, but as time goes by, they have to face various adaptation problems: side effects of medications, medical and social complications, among the latter the return to work, social and family life [12,16,17]. The analysis of quality of life, with its respective components and human responses in patients with a history of CKD is recent. Therefore, the needs inherent in the nursing care process may go unnoticed when directing care for people with these characteristics. Although there are numerous studies that quantitatively address health-related quality of life, [4,18,19] qualitative studies such as the present one provides particular evidence to integrate it into the holistic process of the nursing-patient relationship at different levels of care [13,20]. Therefore, the objective of this study is to analyze the perception of quality of life of people with kidney transplantation and kidney transplant candidates treated at the High Specialty Medical Unit of Mérida, to identify the related human responses through an interpretive phenomenological approach.

Methodology

Design

A qualitative study was conducted with an interpretative phenomenological approach. From this design it is possible to reach the understanding of the experiences and the articulation of similarities and differences in the meanings and human experiences of people with kidney transplantation and candidates for kidney transplantation. Although it is not possible to make generalizations of the results of this study, particular data are reached with transferability to other populations with similar characteristics [6,7,14]. This article followed the COREQ [Consolidated criteria for reporting qualitative research] criteria to enhance its quality and clarity [21].

Study and Sampling Population

An intentional sampling was carried out, obtaining a final sample consisted of 11 people with a history of ERD: 7 candidates to receive kidney transplant and 4 transplanted, who received health services in the High Specialty Medical Unit of Mérida [UMAE] of the Mexican Institute of Social Security [IMSS]during the period from November 2019 to February 2020.

Data Collection

Data were collected through semi-structured interviews conducted during their follow-up consultations. Interviews lasted 30 to 40 minutes, were recorded in audio format, and field notes were taken. Table 1 presents the questions asked during the semistructured interviews.

Table 1: Semi-structured interview questions.

Ethical Considerations

The study respects ethical principles: beneficence, nonmaleficence, justice and autonomy. The research study protocol, with folio R-2018-785-129, was approved by the ethics committee of the High Specialty Medical Unit of the Mexican Social Security Institute. The testimonies presented herein are referenced with codes to safeguard the identity of the participants.

Information Processing

Semi-structured interviews were transcribed verbatim and then analyzed using content analysis. This analysis process consisted of:
1. Encoding the data and establishing a data index.
2. Categorize the content of the data into meaningful categories; and
3. Determine the topics related, in this case human responses, to the previously defined categories [7,22]. In the results section, tables are presented that allow to visualize the categories of analysis delimited in Table 2 from Urzúa and Caqueo [23], the human responses within the categories and, finally, testimonies of the participants; all of the above accompanied by interpretive narrative.

Table 2: Number, crude and age-standardized rate per 100,000 by sex and time during 2005-2014 in the Nghe An province.

Note: * Categories of the concept of quality of life from Urzúa and Caqueo.

Quality Criteria

Once the transcript of the interviews was completed, the 11 participants were asked to verify that the information interpreted was correct. Also the protocolization related to the organization of the data, the detailed and meticulous description of the selection of the sample and the context in which the study is carried out, facilitate the possibility of transfer and reproducibility of the same in similar conditions, thus providing another criterion of qualitative quality.

Results

Characteristics of the participants Years of age were a median of 37 [mean 39]and SD=13 in the 11 participants. In people who were candidates for RT, the median was 37 [mean 41]and in those with RT it was 35.7 years [mean 41), respectively. In the latter group two people were 6 months or less old after receiving RT, one was 1 year old, and one person was 10 years old. Table 3 shows that the majority of the total sample was made up of men who worked as employees.

Table 3: Sociodemographic characteristics of the 11 participants included in the study.

Quality of Life: Perception in Kidney Transplant Candidates

Table 4 shows the interpretations related to the categories: concept of quality of life with their respective domains: physical, economic, family, and social, then the identified human responses are presented. Most of the participants said that quality of life is to be well physically, mentally, and emotionally, as well as to have all the basic services and not depend on renal replacement treatments: dialysis or hemodialysis. In the physical domain, people highlight discomfort, pain and discomfort related to the procedures of renal replacement therapies or the body itself: chronic or bone pain, for example, these human responses largely condition the inability to enter the labor field. In the economic domain, the participants report that they are unable to carry out the activities of any employment due to physical disability, and therefore, consider that their monetary income from a trade or employment is limited, scarce or null. In addition, they stressed that the economic resources are focused on financing the management of the health itself: laboratory tests, transportation, extraordinary treatments, appointments, and medical consultations, among others; these efforts are complicated precisely by the lack of monetary inputs.

Table 4: Quality of life: perception of kidney transplant candidates.

Note: *Categories of the concept of quality of life from Urzúa and Caqueo.

In the family domain, people identify the importance of the support, care, and understanding they receive, received, and expect to receive from their family in the ups and downs related to their state of health and well-being. In this regard, some express feelings of feeling a burden for their relatives for the extra activities that the latter perform in health management, which generates tension and uncertainty. However, the interviewees expressed the motivation generated by their family environment: mothers, children and grandchildren, among other ties, drive the desire to want to get out of their problem and be patients waiting for the transplant. In the emotional domain, each of the people interviewed expressed their affectation at different points that leads them to present low self-esteem: fear, frustration, depression, sadness and uncertainty are some of the emotions they expressed among their testimonies. Participants follow a continuous coping process, because not every day they feel with all the energy and motivation to continue with everyday life. The emotional perception of the interviewees was reflected in their features during the interviews, they touched points that led them to cry, they expressed how difficult it is to live with a dysfunctional organ, the uncertainty before the latent complications that can even make them lose their lives.

Quality of life: Perception in People with Kidney Transplantation

Table 5 shows that most participants consider that quality of life involves physical, environmental and personal well-being as components. For one of the interviewees, it means no longer relying on external factors to sustain life; another considered that the longer he can extend his life is better for the quality of it, considered that discomforts are companions of life. In the physical domain, the interviewees expressed the freedom to perform various activities and eat food without affecting their quality of life. They expressed that they could move and travel without thinking about the need to carry too many supplies related to their treatment. They also stated that they can eat food without causing discomfort or altering their clinical parameters, especially water, which was previously restricted. In the economic domain, participants report that they have time and autonomy to build opportunities for insertion into trades, jobs and vocational or educational training. One case mentioned that the ability to acquire economic resources improves their quality of life, another participant reports that they can work freely without thinking about the times of some renal therapy, finally, a case refers that they returned to normal by fully taking these opportunities that they previously addressed discreetly.

Table 5: Quality of life: perception of kidney transplants.

Note: *Categories of the concept of quality of life from Urzúa and Caqueo.

In the family domain, the perception and feelings of being considered a burden on their families has decreased along with the amount of care related to renal replacement therapies from which transplanted participants are already exempt; people mentioned that despite the constant support of their relatives there was a physical distancing seeking to reduce the crossing of infections, a situation that in recent times has ended and they can share more time and experiences together. In the emotional domain, trust and emotional balance were interpreted in the participants. Two people mentioned that they feel they have a new opportunity before life, to restart it and have new experiences that they previously did not consider possible. Two people referred to the need to have confidence and know how to take the advice of health personnel: doctors and nurses. Finally, one participant described that he was overwhelmed by living a few days in isolation after his transplant, necessary to prevent infections, but at the same time accepts that it is necessary to improve his quality of life.

Discussion

The quality of life of people with a history of renal pathologies is affected since the first clinical manifestations, the QoL in this sector has shown deficiencies, low levels or areas of opportunity with respect to the rest of the population [24]. Physical, environmental and personal well-being are part of the conception of quality of life in people with renal pathologies, whether they have been transplanted or not. In the early stages of the disease there are a series of negative perceptions of the disease and its mediate and immediate quality of life that, ultimately, can influence their coping actions, these perceptions can trigger anxiety, depression, coping, autonomy, self-esteem and accelerated progression of the disease [25]. In the identification of human responses in patients with chronic kidney disease, the main physiological risks related to this pathology have been highlighted. Farias et. al. points out the overstating of biological and complication-related human responses by nursing staff providing care to patients with nephropathies in a renal center. Among 24 diagnostic labels identified, the most frequent were “risk of infection”, “excess fluid volume”, “hypothermia”, among others whose main domains were located in Safety / Protection and Activity / Rest, on the other hand, “low situational self-esteem” was ranked 16th in frequency [26] corresponding to the Self-perception domain in the NANDA-I [20]. The above shows what Spilogon et. al. points out as an area of opportunity in the nursing process because it has the flexibility and openness to consider the perceptions and preferences of the user, in this case of the patient with nephropathies [27].
In the emotional category, low self-esteem was detected in participants with CKD without transplantation, and that is that a patient with CKD has needs for recognition and esteem, so the people in charge of their care should promote favorable behaviors in coping with the pathology and attachment to treatment, avoiding judging and repressing the failures of our human condition [28]. In contrast, participants who had received a kidney transplant manifested confidence and emotional balance, something that could be considered normal after receiving the expected transplant according to Tucker, et. al. [29]. From a quantitative approach Rocha et. Al. point out that the higher the quality of life, the better the assessment of the self-esteem of people with chronic kidney disease after transplantation [30].
In the economic category, while people who had not received kidney transplantation conceived the inability to enter the workforce among their perception of quality of life, those who had received kidney transplantation indicated greater time and autonomy to build job and academic opportunities. Reports indicate that patients with chronic kidney disease face many barriers to staying or joining the workforce after starting dialysis: limited opportunities, lack of financial resources to invest, fatigue and other symptoms of kidney failure, potential loss of disability benefits or medical follow-up, dialysis scheduling, and employer bias. The societal perception that patients with CKD cannot work completes a vicious cycle of low employment expectations [25,31]. In the family category, the perception of “being a burden” for family members influences is an important component in the perception of the quality of life of people with transplantation and without kidney transplantation. Evidence indicates that family members of patients with a history of renal pathologies manifest sleep interruptions, depression, anxiety, among other disorders associated with unforeseen responsibilities related to the treatment and logistics of their relatives; they must also deal with insufficient information, medication regimen and be accompanied by periodic hospitalizations [32]. NANDA International classifies problems into plausible diagnostic labels of interventions focused on promoting the health of individuals, the family. and community, we can mention: Risk of fatigue of the role of caregiver, Fatigue of the role of the caregiver, Dysfunctional family processes, Willingness to improve family processes, among others [20].
In the physical category, participants without kidney transplantation are identified as a condition for quality of life, a common and often severe manifestation in various populations with CKD; with prevalence’s of 40% to 60% is a strong imperative to establish the management of chronic pain as a clinical and research priority [33]. In this regard, the labels acute and chronic pain are available in NANDA-I [20]. Although pain and physical limitation decreases after a kidney transplant, it is important to mention that the physical and nutritional autonomy indicated by the participants of the present can generate an excess of confidence and the acquisition of unhealthy practices. Physical training regulated by physiotherapy specialists appears to be safe in kidney transplant recipients and is associated with improved quality of life and exercise capacity [34]. With respect to diet, the Mediterranean and DASH (Dietary Approaches to Stop Hypertension) diets have been shown to be the most beneficial dietary patterns for the population after kidney transplantation by focusing on less meat and processed foods, while increasing intake of fresh foods and plant-based options. [35]. Knowledge and awareness in the renal transplant population should be a cornerstone of therapy and an integral part of nursing responsibilities. Therefore, nurses should educate patients about self-care behaviors and remind them of the dangerous complications of abandoning them [28].
In participants who had not received a kidney transplant, there was an expectation of receiving a kidney transplant to improve their quality of life and from it to improve their quality of life. In this aspect we can mention the benefits before the expectation of receiving a kidney transplant mentioned by Santos et. al. who in a group of people with Brazilian nephropathies detected that patient who were not waiting for transplantation were at risk of poor quality of life, mainly in the emotional and physical aspects; those who were not awaiting transplantation died more frequently in the next 12 months [36]. However, betting on kidney transplantation to improve the quality of life in patients with nephropathies is not entirely recommended, in this regard we can cite the studies of Schulz et. al. and Smith et. al. published in 2014 and 2019, [29,37] who reported that before transplantation patients can overestimate gains in quality of life without finding significant improvements in quality of life after being transplanted.
Kidney transplantation is not a guarantee of improvement in quality of life in all patients with nephropathies, in the present study, those people who had received the kidney transplant did not consider an absolute improvement in their quality of life. The literature notes that kidney transplants can provide dramatic improvements in quality of life and health status, however, the effects on improvement are not universal and patients live in constant uncertainty as they are aware of the likelihood of graft dysfunction [29]. There are samples that have indicated that the expectation about the functionality or rejection of the graft generates greater fear and uncertainty than death itself [38]. The results on the perception of quality of life in people receiving renal replacement therapy support the trend of the last decade focused on the analysis of this category beyond only assessing life expectancy [39]. The limitations of the present are the risk of bias due to the same interpretative approach and the inability to generalize the results to the study population. To compensate for the above, criteria of methodological rigor were followed and from a particular context the search for generalities was made, reinforcing the results with respect to other studies [21].

Conclusion

In transplant patients, a perception of absolute or discomfortfree quality of life is not achieved and human responses that require care and interventions to achieve the highest level of well-being are still manifested. The construction of the concept of quality of life includes physical, mental, personal and social elements feasible to document and in which to exercise interventions for the benefit of the people treated and their families, it is evident that human responses do not only obey physiological needs.

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Lower Sensitivity of CAT and ER to Melatonin May Lead to Poor Ooplasmic Maturation of Porcine Oocytes from Heat Stress

Introduction

High temperature not only reduces the oocytes maturation quality [1-4] but also has a tremendous adverse impact on the animal ovarian function and embryo attachment [5,6]. Heat stress (HS) damage of oocyte can inhibit cumulus cells expansion [7], which lead to the abnormal distribution of organelles [8,9], decreased antiapoptotic and estrogen receptor gene expressions, enhanced apoptosis gene expression [10], as well as increased Reactive Oxygen Species (ROS) concentration [11,12] in the oocytes. In fact, excessive ROS can induce DNA damage and lipid peroxidation, disrupt the mitochondrial function [13,14], and induce abnormal gene expression and protein synthesis [15]. Reportedly, mitochondrial maturation distribution is an important indicator of the oocyte quality. Oocytes with poor maturation quality have a non-uniform mitochondrial distribution, whereas mitochondria are uniformly distributed in the ooplasm [16]. In order to protect oocytes from HS damage and improve their maturation quality, several materials are supplemented in the maturation medium during in vitro oocyte maturation, such as insulin-like growth factor, β-mercaptoethanol, astaxanthin, anthocyanins, Melatonin (MT), and coagulated proteins [17-20].
It has been reported that MT is involved in regulating several different physiological processes; it can promote the expression of antioxidant-related genes and improve the oocyte maturation quality and embryo developmental potential [21]. In addition, MT concentration of 10-9 M has been proven to be effective in promoting porcine oocyte maturation and development [22]. Oocyte maturation involves several complex events that coordinates nuclear and cytoplasmic maturation processes. Cytoplasmic maturation events following meiotic maturation is much more difficult to assess microscopically, such as the abnormal distribution of the mitochondria, lipid droplets [23], and Glutathione (GSH) concentration detection. However, the nuclear maturation process involves the Germinal Vesicle (GV) breakdown, chromosomal arrangement, and completion of Metaphase 1 (MI) by extruding the first polar body into the perivitelline space of the oocytes, all of which were observed by stereomicroscopy in a previous study [24]. It has been reported that HS can increase the expression of proapoptotic genes, enhance the activity of caspase proteins, and trigger the apoptosis pathway [25,26]. The members of the BCL- 2 family play a key role in regulating apoptosis, among which the expressions of the proapoptotic gene BAX and antiapoptotic gene BCL-2 as well as the ratio of these two gene expression levels are generally deemed as indicators for predicting the oocyte maturation quality and the embryo developmental potential.
Past studies in cattle have reported that the expressions of genes related to oocyte maturation quality and developmental potential were greatly reduced after the Cumulus and Oocyte Complexes (COCs) were subjected to HS at 41℃ for 12h. The present study discusses the sensitivity of BCL-2, BAX, CAT, and ER to high temperature and MT and analyzes the relationship among their sensitive differences and porcine oocyte maturation quality and developmental potential in vitro. We first discovered that the low sensitivity of CAT and ER to MT possibly contributes to the strong relationship with the poor porcine oocyte maturation quality and the developmental capacity of the embryos in vitro. We believe that the present results would be helpful in enhancing oocytes utilization and would provide a practical guide toward improving pig fertility during the high-temperature season.

Materials & Methods

All reagents used in this experiment were purchased from Sigma Chemicals (St. Louis, MO, USA), unless otherwise specified.

Oocytes Collection and Culturing In Vitro

The ovaries were acquired from a slaughterhouse and dispatched to our laboratory within 2 h of collection in a thermos flask containing sterile saline at 35-37℃. The COCs were extracted from follicles (of 2-6-mm diameter) with a disposable syringe (10 mL; No. 18 needle), and only COCs with uniform ooplasm and compact cumulus cells were maturation cultured in an incubator with 5% CO2 and 95% humidified air atmosphere, as follows: some COCs were cultured at 38.5℃ for 42 h in a maturation medium (Control, No HS); some COCs were cultured at 41.5℃ for 4 h and then transferred for continuous culturing at 38.5℃ for 38 h in the maturation medium (HS group); and the other COCs were cultured at 41.5℃ for 4 h in a medium supplemented with 10-9 M MT, after which it was subjected to continuous cultured for 38 h in a maturation medium at 38.5℃ (HSMT group). The maturation medium consisted of TCM199 (with Earle’s Salts; Gibco, Grand Island, NY, USA) supplemented with 10% porcine follicular fluid (PFF), 0.1 mg/mL cysteine, 0.065 mg/mL penicillin, 10 ng/ mL epidermal growth factor (EGF), 10 IU/mL equine chorionic gonadotropin (eCG; Intervet Pty. Ltd., Boxrneer, Australia), and 10 IU/mL human chorionic gonadotrophin (hCG; Intervet Pty. Ltd.). All the experiments were repeated thrice.

Assessment of the First Polar Body Expulsion Rate

The COCs from different groups were respectively stripped off cumulus cells by gentle pipetting in Phosphate-Buffered Saline (PBS) supplemented with 0.1% hyaluronidase, and the first polar body expulsion rate was determined under a stereomicroscope. A total of 150 denuded oocytes from each group were used for determining the rate of the first polar body expulsion. All oocytes used in the subsequent experiments had their first polar body expulsed.

Ooplasmic ROS Detection

ROS was detected using the Reactive Oxygen Species Assay Kit (S0033; Beyotime®, Haimen, Jiangsu, China) as per the manufacturer’s instructions. Briefly, 50 matured oocytes from each group were rinsed in the PBS solution thrice, followed by dying with 10× M ROS dye in the dark for 10 min. Next, photographs were taken under a fluorescence microscope (TE2000-s; Nikon, Japan). The fluorescence intensity analysis was performed with the Image J (Version 1.8.0) software, and the experiment was repeated thrice.

Ooplasmic Mitochondrial Distribution Analysis

Mitochondrial staining was performed with the Mito-Tracker Red CMXROS (C1049; Beyotime®) as per the manufacturer’s instructions. Briefly, 50 oocytes from each group were stained with 200-nM mitochondrial dye in the PBS solution for 25 min at 37℃ in the dark after washing in PBS thrice. Then, the stained oocytes were observed under the fluorescence microscope. The mitochondrial distribution pattern of porcine oocytes was then characterized based on two main distribution features: uniform distribution throughout the ooplasm or non-uniform distribution throughout the ooplasm.

Parthenote Production and Culture In Vitro

A total of 100 matured oocytes from each group were transferred to the activation medium (composed of 1.0 mM CaCl2, 0.1 mM Mg Cl2, 0.3 M mannitol, and 0.5 mM HEPES). Matured oocytes were activated with two pulses of 120 V/mm DC for 60 ms with the Electro-Cell Manipulator BTX 2001 (BTX Inc., USA). After activation, the parthenotes were subsequently cultured in 2 mM 6-dymethylaminopurine (6-DAMP) for 6 h and then the parthenotes were transferred into the PZM-3 medium for 7 days in an incubator at 39℃ under 5% CO2 atmosphere in humidified air. The rates of cleavage and blastocyst transfer were observed respectively on days 2 and 7 after oocytes activation.

Blastocyst Cells Staining

A total of 10 blastocysts were randomly selected from each group and fixed in 4% paraformaldehyde prepared in PBS containing 0.1% polyvinyl alcohol (PVA) for 1 h after washing thrice, and the blastocysts were then permeabilized in PBS-0.1% PVA solution containing 0.3% Triton for 30 min. After washing, the blastocysts were transferred to a solution supplemented with 10 μg/mL DAPI dye for 1 min, and then the blastocysts were mounted on a slide and covered with a coverslip. The total blastocyst count in each group was counted under the fluorescence microscope.

Gene mRNA Expression

The expressions of BCL-2, BAX, CAT, and ER were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA of 100 oocytes from each group was extracted by using the Micro RNA Extraction Kit (160027349; Qiagen, CA, USA) as per the manufacturer’s instruction. After washing in PBS, the oocytes were transferred to a 200-μL centrifuge tube for pre-cooling and then stored at -80℃ for further processing. After total RNA extraction, cDNA synthesis was performed for 30 min at 55°C using the Omniscript Reverse Transcription Kit (Invitrogen) with oligodT primer. PCR was performed by using the Maxime PCR Premix with SYBR Green (TaKaRa Bio Inc., Otsu, Japan) supplemented with each primers and cDNA samples under the following conditions: predenaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 56°C for 30 s, elongation at 72°C for 30 s, and a final extension at 72°C for 5 min for 40 cycles using the Eppendorf Mastercycler (Eppendorf, Hamburg, Germany). According to the mRNA sequences of Sus scrofa genes published on Gen Bank, we designed primers with the Primer 5.0 software and synthesized by Shanghai Bioengineering Co., Ltd. (Shanghai, China). The primers used in the present study were verified for their availability by RTPCR. Real-time quantitative PCR was performed by the comparative Ct (2 -△△Ct ) method, and the results obtained for each gene in each cDNA pool were normalized based on the GAPDH ratio. The primers and Genebank source accessions for each gene are listed in Table 1.

Table 1: RT-PCR primers and Genebank source.

Statistics

The percent values were subjected to log transformation before analysis, and the quantitative data were analyzed by least-square analysis of variance (ANOVA) using the General Linear Models (GLM) procedures of the Statistical Analysis System (SAS Institute, Cary, NC, USA). We corrected the real-time PCR data by using the GAPDH data as a covariate for the analysis of differences. All data were expressed as mean ± SEM, with P < 0.05 deemed as statistically significant. All experiments were repeated thrice.

Results

Assessment of the First Polar Body Expulsion Rates of Oocytes in Different Groups

The first polar body expulsion rates of porcine oocytes are given in Table 2. Although the first polar body expulsion rate greatly decreased in the HS group in comparison with that in the control group, the corresponding rate in the HSMT group significantly increased again but exhibited no significant difference with that in the control group (P > 0.05). The first polar body of porcine oocytes was observed under a stereomicroscope (Figure 1A), and the observation was confirmed under a fluorescence microscope (Figure 1B). Figure 1C represents oocytes without the expulsed first polar body, indicating that fluorescence occurred only at the nuclear sites observed under the fluorescence microscope.

Table 2: The porcine first polar body expulsion rates in different groups.

Figure 1: Porcine oocytes with or without the first polar body expulsed
A. The stereomicroscopic examination of the matured oocytes with the first polar body expulsed, as pointed by the arrows.
B. The matured oocytes were stained with fluorescent dye Hoechst 33342, where both the polar body and the nucleus exhibited fluorescence.
C. Oocytes with no polar body expulsed were stained with fluorescent dye Hoechst 33342, where only the nucleus exhibited fluorescence as pointed by the arrows. Bar=100 μm.

ROS Concentration in Oocytes of Different Groups

The ROS concentrations in the HS group was significantly increased relative to those in the control group. In addition, although the ROS concentration in the HSMT group decreased significantly, it remained significantly higher than that in the control group as showed in Table 3 (P < 0.05). The proportion of oocytes with uniform mitochondrial distribution in porcine oocytes of different groups. Figure 2 supports that the proportion of oocytes with a uniform mitochondrial distribution in the HS group was significantly lower than that in the control group (P < 0.05). Although the proportion of oocytes with uniform mitochondrial distribution was greatly enhanced in the HSMT group, it remained significantly lower than that in the control group (P < 0.05). Figure 3 represents the uniform and non-uniform mitochondrial maturation distribution in the ooplasm.

Figure 2: The proportion of oocytes with better mitochondrial maturation distribution in different groups. Different letters (a-c) over a bar means significant difference (P < 0.05). Each experiment was repeated three times.

Figure 3: The condition of mitochondrial maturation distribution in ooplasm Oocyts were fluorescent stained by Mito-Tracker Red, and A. Represents the oocyte of better maturation quality with even distribution of mitochondria in ooplasm. B. Represents the oocyte of poor maturation quality with uneven distribution of mitochondria in ooplasm. Bar = 100 μm.

Table 3: ROS contents of oocytes in different groups.

Assessment of Porcine Oocytes Developmental Potential In Vitro

As can be seen in Table 4, the cleavage rate, blastocyst rate, and the total number of blastocysts were significantly lower in the HS group than in the control group (P < 0.05). As compared with those in the HS group, the rates of cleavage, blastocysts, and the total number of blastocysts in the HSMT group were significantly increased (P < 0.05), with no significant difference in the cleavage rate relative to that in the control group (P > 0.05). Nevertheless, the blastocyst rate and the total number of blastocysts remained significantly lower than the respective control values (P < 0.05). The results of blastocyst cells staining are depicted in Figure 4.

The mRNA Expressions of CAT, BCL-2, BAX, and ER in Oocytes of Different Groups

As shown in Figure 5, the mRNA expressions of CAT, ER, and BCL- 2 were decreased, whereas the BAX expression was significantly increased in the HS group in comparison with those in the control group. However, in the HSMT group, the mRNA expression levels of BCL-2 and BAX were restored to the control levels, while the CAT and ER expressions remained significantly lower than the control values (P < 0.05). Moreover, no significant difference was noted in the mRNA expression of ER between the HS and HSMT groups (P > 0.05).

Figure 4: The fluorescence staining of blastocyst cells. The blastocyst cells was fluorescent stained with 10μg/ml DAPI dye and a number of 10 blastocysts randomly selected from each group.
A. The blastocyst comes from the Control
B. The blastocyst comes from the HS group
C. The blastocyst comes from the HSMT group. Bar = 100 μm.

Figure 5: The mRNA expressions of CAT, BCL-2, BAX and ER genes in oocytes of different groups. Different letters (a-c) over a bar means significant difference (P < 0.05). Each experiment was repeated three times.

Table 4: The porcine oocytes developmental potential in different groups.

Discussion

Previous studies have shown that the effects of MT exposure on protecting oocytes from HS damage varied among different animals. Negrón-Pérez reviewed that the cleavage rate and the total number of blastocysts in dairy cows increased when MT was supplemented in the HS system [27], while another bovine study demonstrated that MT has no significant effect on the cleavage rate and on the total number of blastocysts [28]. The present study on porcine suggested that, although 10-9 M concentration of MT could significantly improve the cleavage rate, the blastocyst rate and total cell of blastocysts remained significantly lower in the HSMT group than in the control group (P < 0.05). Indeed, neither previous bovine studies nor the present porcine study indicated whether high-concentration MT supplementation could increase the total cell of blastocysts. With regards to porcine oocyte maturation, the ooplasmic maturation is usually evaluated by some molecular events, bioreaction, or organelle distribution [29], and the nuclear maturation is estimated by the first polar body expulsed into the perivitelline space of oocytes [30].
The results of the present study indicated that the first polar body expulsion rate was greatly increased in the HSMT group in comparison with that in the HS group, exhibiting no significant difference relative to that in the control group. We thus speculated that supplementation with 10 IU/mL Equine Chorionic Gonadotropin (eCG) and 10 IU/mL human Chorionic Gonadotrophin (hCG) to the maturation medium was sufficient for porcine oocyte nuclear maturation in the present experiments, considering that gonadotropins are responsible for the resumption of oocyte meiosis and for the promotion of nuclear maturation by the cAMP/PKA/MAP kinase pathway [31]. Although MT supplementation could significantly increase the ratio of oocytes with a uniform mitochondrial distribution, it remained significantly lower in the HSMT group than in the control group. Several other researches have demonstrated that HS could influence abnormal mitochondrial distribution, which is detrimental to oocyte maturation and development in vitro [32-37]. The data from production also suggests that the ooplasmic mitochondrial distribution was poor during the summers, but it improved during the autumn season [38].
The poor mitochondrial maturation in the HSMT group oocytes may attribute to the higher intracellular ROS concentration, considering that ROS is one of the main factors that result in diverse damages to oocyte maturation and development [39-42]. The present experiments demonstrated that 10-9 M MT concentration is insufficient to eliminate the excessive ROS in the oocytes of the HSMT group. Our study is the first to indicate that the BAX and BCL-2 were sensitive to both high temperature and MT. As for the CAT, 10-9 M concentration of MT could not restore its expression to the control level, which may explain the higher ROS concentrations in the HSMT group. Estrogen receptor gene is closely associated with the fertility of female animals and follicular development and oocyte maturation [43-45]. In our study, we found that the ER was sensitive to high temperature, but extremely insensitive to MT exposure. The reduction in ER expression induced the lack of binding sites for estrogen, which lowers the fertility of female animals under hot conditions [46]. Recent data in production also confirmed that the fertility of sow was lower in the summer season than in other seasons [47]; moreover, past studies in cows have reported that subcutaneous implantation with 18 mg MT could only partially alleviate the adverse effects of HS on the reproductive performance of cows in the hot season [48-50].
Backed by our research, we propose that the reduced secretion of MT and the insensitivity of ER to MT is most probably related with the lower fertility of animals in high temperature season. However, until date, the quantity of MT required to counteract the adverse effects of HS on pig remain unreported. Based on our experiments, we believe that much more than the 10-9 M concentration of exogenous MT would be required. The present experimental results imply that the lower fertility of animals in high temperature season can be attributed to the sensitive differences in the expressions of BCL-2, BAX, CAT, and ER to high temperature and MT, which provides important insights to the application of MT toward improving the fertility of animals during the high temperature season.

Conclusion

BCL-2 and BAX were found to be sensitive to both high temperature and MT exposure; however, CAT and ER, especially the latter, were found to be sensitive only to high temperature and extremely insensitive to MT exposure. The sensitive differences in these genes contributed to poor ooplasmic transfer, lack of nuclear material transfer, and maturation, which further hampered the developmental capacity of porcine oocytes.

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