Topic:Infertility

Expression pattern of germ cell markers in cryptorchid stallion testes.
Reproduction in domestic animals = Zuchthygiene    April 13, 2024   Volume 59, Issue 4 e14561 doi: 10.1111/rda.14561
Shakeel M, Choi Y, Yoon M.Cryptorchidism affects spermatogenesis and testis development, often resulting in stallion subfertility/infertility. This study aims to identify the specific germ cells impacted by cryptorchism in stallions. In a previous study, we found that PGP9.5 and VASA are molecular markers expressed in different germ cells within stallions. Herein, we assessed the heat stress-induced response of spermatogonial stem cells (SSCs) in the seminiferous tubules (ST) of cryptorchid stallion testes (CST) and normal stallion testes (NST). This goal was accomplished by comparing PGP9.5 and VASA expression pattern...
Phospholipase C Zeta 1 (PLCZ1): The Function and Potential for Fertility Assessment and In Vitro Embryo Production in Cattle and Horses.
Veterinary sciences    December 11, 2023   Volume 10, Issue 12 698 doi: 10.3390/vetsci10120698
Gonzalez-Castro RA, Carnevale EM.Phospholipase C Zeta 1 (PLCZ1) is considered a major sperm-borne oocyte activation factor. After gamete fusion, PLCZ1 triggers calcium oscillations in the oocyte, resulting in oocyte activation. In assisted fertilization, oocyte activation failure is a major cause of low fertility. Most cases of oocyte activation failures in humans related to male infertility are associated with gene mutations and/or altered PLCZ1. Consequently, PLCZ1 evaluation could be an effective diagnostic marker and predictor of sperm fertilizing potential for in vivo and in vitro embryo production. The characterization ...
Intratesticular transplantation of allogenic mesenchymal stem cells mitigates testicular destruction after induced heat stress in Miniature-horse stallions.
Journal of equine veterinary science    November 3, 2023   Volume 132 104961 doi: 10.1016/j.jevs.2023.104961
Testicular degeneration (TD) is the most frequent cause of sub or infertility in stallions. Currently, mesenchymal stem cells (MSC) have been studied as a therapeutic option for several diseases including induced-TD in laboratory animals. Therefore, this study aimed to evaluate the effect of intratesticular MSC therapy on the testicular histology of stallions submitted to scrotal heat stress. Ten healthy Miniature-horse stallions were submitted to testicular heat stress induced by a heating wrap device (42-45°C). Afterward, the stallions were divided into two groups and treated seven days lat...
Sperm Quality Assessment in Stallions: How to Choose Relevant Assays to Answer Clinical Questions.
Animals : an open access journal from MDPI    October 6, 2023   Volume 13, Issue 19 3123 doi: 10.3390/ani13193123
Egyptien S, Deleuze S, Ledeck J, Ponthier J.Stallion sperm analysis is indicated for infertility diagnosis, pre-sale expertise, production of fresh or frozen doses, and frozen straw quality control. Various collection methods are described, and numerous assays can be performed on semen. Determining an approach for each of these cases is challenging. This review aims to discuss how to obtain relevant clinical results, answering stallion owners' concerns. Semen can be collected with an artificial vagina on a phantom or a mare, by electro-ejaculation under anesthesia, or after pharmacological induction. The collection method influences the...
The potential role of miRNAs and regulation of their expression in the development of mare endometrial fibrosis.
Scientific reports    September 24, 2023   Volume 13, Issue 1 15938 doi: 10.1038/s41598-023-42149-3
Wu00f3jtowicz A, Molcan T, Lukasik K, u017bebrowska E, Pawlina-Tyszko K, Gurgul A, Szmatou0142a T, Bugno-Poniewierska M, Ferreira-Dias G....Mare endometrial fibrosis (endometrosis), is one of the main causes of equine infertility. Despite the high prevalence, both ethology, pathogenesis and the nature of its progression remain poorly understood. Recent studies have shown that microRNAs (miRNAs) are important regulators in multiple cellular processes and functions under physiological and pathological circumstances. In this article, we reported changes in miRNA expression at different stages of endometrosis and the effect of transforming growth factor (TGF)-β1 on the expression of the most dysregulated miRNAs. We identified 1, 26, ...
MicroRNAs in equine Endometritis: A review of pathophysiology and molecular insights for diagnostic and therapeutic strategies.
International immunopharmacology    September 17, 2023   Volume 124, Issue Pt B 110949 doi: 10.1016/j.intimp.2023.110949
Asif S, Umar T, Umar Z, Jamil H, Feng H, Zhang P, Umer S.Endometritis plays an important role in mare infertility. Certain infectious agents interfere with the innate immune system of endometrium, causing a systemic inflammatory response that lasts for a long time and circulates via the blood or cellular degeneration, leading to endometritis due to bacterial endotoxins. Different small, non-coding RNA molecules are involved in many biological functions. For instance, microRNAs (miRNAs) are involved in the post-transcriptional regulation of gene expression. These miRNAs are important regulators of gene expression, primarily via inhibiting transcripti...
Heat stress and stallion fertility.
Journal of animal science and technology    July 30, 2023   Volume 65, Issue 4 683-697 doi: 10.5187/jast.2023.e29
Shakeel M, Yoon M.The threat posed by increased surface temperatures worldwide has attracted the attention of researchers to the reaction of animals to heat stress. Spermatogenesis in animals such as stallions is a temperature-dependent process, ideally occurring at temperatures slightly below the core body temperature. Thus, proper thermoregulation is essential, especially because stallion spermatogenesis and the resulting spermatozoa are negatively affected by increased testicular temperature. Consequently, the failure of thermoregulation resulting in heat stress may diminish sperm quality and increase the li...
Temporal trends in equine sperm progressive motility: a systematic review and meta-regression.
Reproduction (Cambridge, England)    May 2, 2023   Volume 165, Issue 6 M1-M10 doi: 10.1530/REP-22-0490
Harris IT, Maddock C, Farnworth M, Nankervis K, Perrett J, Pyatt AZ, Blanchard RN.Adverse trends in reproductive function are a concern in humans, companion, livestock, and wildlife species. This study indicates that equine populations are at risk of a comparable decline in sperm progressive motility. There is increasing evidence reporting geographically sensitive adverse trends in human semen quality, with parallel trends observed in the dog sentinel. Despite significant economic and welfare complications associated with poor testicular function, trends in current equine populations are undetermined. Given the predictive value of sperm progressive motility (PMOT) in male f...
Case report: Long term follow-up of a large unilateral epididymal cyst in a stallion used for teaching: Is this condition associated with infertility?
Frontiers in veterinary science    March 29, 2023   Volume 10 1145742 doi: 10.3389/fvets.2023.1145742
Silva GF, Cunha R, Carvalho F, Ribeiro M, Rocha A, Amorim I, Guimaru00e3es T.A 30-year-old Lusitano stallion presented with an enlarged right epididymis. The ultrasound scan revealed a cyst-like formation and the histopathological examination was compatible with epididymal cyst located at the body/tail transition, epididymal spermatocele and sperm granuloma and epididymitis. However, these conditions did not seem to affect the animal's reproductive performance, nor did the semen parameters analyzed over the 8 years after the diagnosis show significant changes. Nevertheless, since the ejaculate contains mostly sperm cells from the tail of the epididymis, where fertile s...
Effect of different adjuvant formulations on the antibody response of horses to porcine zona pellucida proteins.
Veterinary immunology and immunopathology    October 28, 2022   Volume 253 110507 doi: 10.1016/j.vetimm.2022.110507
AbdelKhalek A, Ostafe R, Olave C, HogenEsch H, Turner JW.Immunization with porcine zona pellucida (PZP) proteins is being used successfully to induce infertility in wildlife including horses. However, widespread adoption of this method to control the growth of horse populations requires further refinement in order to induce long-term infertility, reduce the frequency and severity of injection site reactions, and make the vaccines easier to administer. The next generation of PZP-based vaccines will likely be a controlled-release formulation with different adjuvants from the Freund's adjuvants used in existing vaccines. We evaluated the response of eq...
Comparison Between Gynecological Examination Methods and Sample Collection Techniques for the Diagnosis of Endometritis in Subfertile Mares.
Journal of equine veterinary science    October 23, 2022   Volume 119 104147 doi: 10.1016/j.jevs.2022.104147
Teixeira-Soares CM, Viana AGA, Ribeiro IM, Silva KDP, Sancler-Silva YFR, Machado-Neves M.Endometritis is a relevant cause of subfertility in mares. However, the accurate diagnosis, essential for effective treatment, can be difficult due to the variability of results and interpretations resulting from different examination methods and sample collection techniques. The present work compared gynecological evaluation methods and sample collection techniques to diagnose endometritis in subfertile mares. Forty animals with a history of subfertility were selected for gynecological evaluation using clinical methodologies, such as perineal conformation, transrectal palpation and ultrasonog...
Copy number variation of horse Y chromosome genes in normal equine populations and in horses with abnormal sex development and subfertility: relationship of copy number variations with Y haplogroups.
G3 (Bethesda, Md.)    October 14, 2022   Volume 12, Issue 12 jkac278 doi: 10.1093/g3journal/jkac278
Castaneda C, Radoviu0107 L, Felkel S, Juras R, Davis BW, Cothran EG, Wallner B, Raudsepp T.Structural rearrangements like copy number variations in the male-specific Y chromosome have been associated with male fertility phenotypes in human and mouse but have been sparsely studied in other mammalian species. Here, we designed digital droplet PCR assays for 7 horse male-specific Y chromosome multicopy genes and SRY and evaluated their absolute copy numbers in 209 normal male horses of 22 breeds, 73 XY horses with disorders of sex development and/or infertility, 5 Przewalski's horses and 2 kulans. This established baseline copy number for these genes in horses. The TSPY gene showed the...
Seasonal changes in the expression of molecular markers of stallion germ cells.
Journal of equine veterinary science    August 24, 2022   Volume 118 104109 doi: 10.1016/j.jevs.2022.104109
Shakeel M, Jung H, Yoon D, Yoon M.The economic impacts of infertility and subfertility of stallions greatly influence the horse breeding industry. Self-renewal and differentiation of spermatogonial stem cells are the initial processes to maintain an adequate sperm population. Thus, understanding these processes may provide useful information to reveal the causes and remedies of subfertile and infertile stallions. Stallions are seasonal breeders. About 50% of the sperm population is reduced during the non-breeding season (NBS) in stallions. The seasonal regulation of spermatogenesis renders stallions as ideal models to understa...
Metallopeptidades 2 and 9 genes epigenetically modulate equine endometrial fibrosis.
Frontiers in veterinary science    August 12, 2022   Volume 9 970003 doi: 10.3389/fvets.2022.970003
Alpoim-Moreira J, Fernandes C, Pimenta J, Bliebernicht M, Rebordão MR, Castelo-Branco P, Szóstek-Mioduchowska A, Skarzynski DJ, Ferreira-Dias G.Endometrium type I (COL1) and III (COL3) collagen accumulation, periglandular fibrosis and mare infertility characterize endometrosis. Metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) are involved in collagen turnover. Since epigenetic changes may control fibroproliferative diseases, we hypothesized that epigenetic mechanisms could modulate equine endometrosis. Epigenetic changes can be reversed and therefore extremely promising for therapeutic use. Methylation pattern analysis of a particular gene zone is used to detect epigenetic changes. DNA...
Endometrial and vaginal microbiome in donkeys with and without clinical endometritis.
Frontiers in microbiology    August 1, 2022   Volume 13 884574 doi: 10.3389/fmicb.2022.884574
Li J, Zhu Y, Mi J, Zhao Y, Holyoak GR, Yi Z, Wu R, Wang Z, Zeng S.Endometrial and vaginal microbiomes are critical in the study of endometritis, which is an important cause of infertility in donkeys. Our objective was to investigate the difference of the endometrial and vaginal microbiomes between healthy donkey jennies (group C) and jennies with endometritis (group E). Endometrial and vaginal swab samples were collected, and the 16 s rRNA gene amplicon high-throughput sequencing technique was applied to identify the microbial composition in the samples. A similar microbial composition pattern was found between endometrial and vaginal samples, which indica...
Collagen Type III as a Possible Blood Biomarker of Fibrosis in Equine Endometrium.
Animals : an open access journal from MDPI    July 21, 2022   Volume 12, Issue 14 1854 doi: 10.3390/ani12141854
Alpoim-Moreira J, Fernandes C, Rebordu00e3o MR, Costa AL, Bliebernicht M, Nunes T, Szu00f3stek-Mioduchowska A, Skarzynski DJ, Ferreira-Dias G.Collagen pathological deposition in equine endometrium (endometrosis) is responsible for infertility. Kenney and Doig's endometrial biopsy histopathological classification is the gold standard method for endometrosis evaluation, whereby blood biomarkers identification would be less invasive and could provide additional information regarding endometrosis diagnosis and fertility prognosis. This study aimed to identify blood biomarkers for endometrosis diagnosis (42 mares were used in experiment 1), and fertility assessment (50 mares were used in experiment 2). Reproductive examination, endometri...
The Stallion Spermatozoa: A Valuable Model to Help Understand the Interplay Between Metabolism and Redox (De)regulation in Sperm Cells.
Antioxidants & redox signaling    April 11, 2022   Volume 37, Issue 7-9 521-537 doi: 10.1089/ars.2021.0092
Peu00f1a FJ, O'Flaherty C, Ortiz Rodru00edguez JM, Martu00edn Cano FE, Gaitskell-Phillips G, Gil MC, Ortega Ferrusola C. Proper functionality of the spermatozoa depends on the tight regulation of their redox status; at the same time these cells are highly energy demanding and in the energetic metabolism, principally in the electron transport chain in the mitochondria, reactive oxygen species are continuously produced, in addition to that observed in the Krebs cycle and during the β-oxidation of fatty acids. In addition, in glycolysis, elimination of phosphate groups from glyceraldehyde 3-phosphate and dihydroxyacetone phosphate results in the byproducts glyoxal (G) and methylglyoxal (MG); these products are 2...
Evidence-Based Application of Acupuncture in Theriogenology.
Veterinary sciences    January 28, 2022   Volume 9, Issue 2 53 doi: 10.3390/vetsci9020053
Holyoak GR, Ma A.Historical evidence of acupuncture predates written history. It has been a component of Traditional Chinese veterinary medicine for many generations and is officially recognized in recorded history for treating equine disease in the Zhou Dynasty, circa 1050 BC. Drawing from a range of searchable databases, we present the use of veterinary acupuncture related to theriogenology. We touch on human-based medicine only as an introduction to current uses within veterinary medical acupuncture. This review is confined to the use of acupuncture encompassing dry needle, electroacupuncture, aquapuncture,...
Collagen and Microvascularization in Placentas From Young and Older Mares.
Frontiers in veterinary science    January 4, 2022   Volume 8 772658 doi: 10.3389/fvets.2021.772658
Neto da Silva AC, Costa AL, Teixeira A, Alpoim-Moreira J, Fernandes C, Fradinho MJ, Rebordu00e3o MR, Silva E, Ferreira da Silva J, Bliebernicht M....In older mares, increasing collagen fibers (fibrosis) in the endometrium and oviduct predisposes to sub-fertility and infertility. In this study, (i) gene transcription of collagen (qPCR: ); (ii) total collagen protein (hydroxyproline); (iii) collagen distribution (Picrosirius red staining; polarized light microscopy); and (iv) microvascular density (Periodic acid-Schiff staining), were evaluated in mares' placenta, and related to mares age, and placenta and neonate weights. Samples were collected from the gravid horn, non-gravid horn, and body of the placenta from younger ( = 7), and older ma...
Molecular Cytogenetic and Y Copy Number Analysis of a Reciprocal ECAY-ECA13 Translocation in a Stallion with Complete Meiotic Arrest.
Genes    November 26, 2021   Volume 12, Issue 12 1892 doi: 10.3390/genes12121892
Castaneda C, Ruiz AJ, Tibary A, Raudsepp T.We present a detailed molecular cytogenetic analysis of a reciprocal translocation between horse (ECA) chromosomes Y and 13 in a Friesian stallion with complete meiotic arrest and azoospermia. We use dual-color fluorescence in situ hybridization with select ECAY and ECA13 markers and show that the translocation breakpoint in ECAY is in the multicopy region and in ECA13, at the centromere. One resulting derivative chromosome, Y;13p, comprises of ECAY heterochromatin ( array), a small single copy and partial Y multicopy region, and ECA13p. Another derivative chromosome 13q;Y comprises of ECA13q ...
The Mare: A Pertinent Model for Human Assisted Reproductive Technologies?
Animals : an open access journal from MDPI    August 4, 2021   Volume 11, Issue 8 2304 doi: 10.3390/ani11082304
Benammar A, Derisoud E, Vialard F, Palmer E, Ayoubi JM, Poulain M, Chavatte-Palmer P.Although there are large differences between horses and humans for reproductive anatomy, follicular dynamics, mono-ovulation, and embryo development kinetics until the blastocyst stage are similar. In contrast to humans, however, horses are seasonal animals and do not have a menstrual cycle. Moreover, horse implantation takes place 30 days later than in humans. In terms of artificial reproduction techniques (ART), oocytes are generally matured in vitro in horses because ovarian stimulation remains inefficient. This allows the collection of oocytes without hormonal treatments. In humans, in viv...
Andrological and cytogenetic investigations of an infertile Przewalski’s stallion.
Acta veterinaria Hungarica    July 22, 2021   Volume 69, Issue 2 189-193 doi: 10.1556/004.2021.00027
Ku00fatvu00f6lgyi G, Brabender K, Andersson M, Javkhlan A, Nagy S, Pu00e1ble T, Egerszegi I, Hidas A, Sou00f3s I, Kovu00e1cs A.The case of an 8-year-old, sexually active but infertile Przewalski's stallion (Equus ferus przewalskii) was studied. Besides the infertility, the stallion also showed permanent problems with its body condition, being obviously weaker than all the other group members. The horse was kept in a separate place for two years with 12 mares in its harem group (six mares had foals earlier); however, none of the mares covered got pregnant. Andrological and cytogenetic investigations revealed underdeveloped testes, arrested spermatogenesis, azoospermia, and XY/XXY/X0 mosaicism. We classify the case as a...
An integrated overview on the regulation of sperm metabolism (glycolysis-Krebs cycle-oxidative phosphorylation).
Animal reproduction science    July 14, 2021   Volume 246 106805 doi: 10.1016/j.anireprosci.2021.106805
Peu00f1a FJ, Ortiz-Rodru00edguez JM, Gaitskell-Phillips GL, Gil MC, Ortega-Ferrusola C, Martu00edn-Cano FE.An overview of the sperm metabolism is presented; using the stallion as a model we review glycolysis, Krebs Cycle and oxidative phosphorylation, paying special attention to the interactions among them. In addition, metabolism implies a series of coordinated oxidation-reduction reactions and in the course of these reactions reactive oxygen species (ROS) and reactive oxoaldehydes are produced ; the electron transport chain (ETC) in the mitochondria is the main source of the anion superoxide and hydrogen peroxide, while glycolysis produces 2-oxoaldehydes such as methylglyoxal as byproducts; due t...
Endometrial angiogenesis induced by uterine insufflation with an oxygen-ozone gas mixture in mares.
Reproduction in domestic animals = Zuchthygiene    June 3, 2021   Volume 56, Issue 9 1176-1183 doi: 10.1111/rda.13958
Camargo Ferreira J, Silva Amaral R, Mara Cardoso L, Panelli Marchiu00f3 S, Aldrovani Rodrigues M, Fonseca Alves CE, de Meira C.Ozone (O ) therapy has been used to improve peripheral tissue oxygenation in humans and domestic animals. The goal of the present study was to characterize histological changes in the endometria of healthy equines following tissue exposure to gas mixtures enriched with different concentrations of O . Cycling mares without endometrial degeneration were divided into three groups according to treatment (n = 9 mares/group). The uteri from the O , ½O and control groups were insufflated for 3 min with gas containing 42, 21 and 0 μg O ml , respectively. Treatments were performed every three da...
Relationship between endogenous plasma adrenocorticotropic hormone concentration and reproductive performance in Thoroughbred broodmares.
Journal of veterinary internal medicine    May 24, 2021   Volume 35, Issue 4 2002-2008 doi: 10.1111/jvim.16145
Tsuchiya T, Noda R, Ikeda H, Maeda M, Sato F.Pituitary pars intermedia dysfunction (PPID) is an endocrine disorder the clinical signs of which include infertility, but few reports have examined the relationship between PPID and reproductive performance in broodmares. Objective: Broodmares with infertility of unknown etiology were screened for PPID using baseline endogenous plasma ACTH concentrations, and its relationship with subsequent reproductive performance was analyzed. Methods: Sixty-seven Thoroughbred broodmares. Methods: Adrenocorticotropic hormone concentrations were measured in autumn. The broodmares were divided into 3 groups ...
A comparative analysis of the intrauterine transcriptome in fertile and subfertile mares using cytobrush sampling.
BMC genomics    May 22, 2021   Volume 22, Issue 1 377 doi: 10.1186/s12864-021-07701-3
Weber KS, Wagener K, Blanco M, Bauersachs S, Bollwein H.Subfertility is a major problem in modern horse breeding. Especially, mares without clinical signs of reproductive diseases, without known uterine pathogens and no evidence of inflammation but not becoming pregnant after several breeding attempts are challenging for veterinarians. To obtain new insights into the cause of these fertility problems and aiming at improving diagnosis of subfertile mares, a comparative analysis of the intrauterine transcriptome in subfertile and fertile mares was performed. Uterine cytobrush samples were collected during estrus from 57 mares without clinical signs o...
Screening and detection of chromosomal copy number alterations in the domestic horse using SNP-array genotyping data.
Animal genetics    May 19, 2021   Volume 52, Issue 4 431-439 doi: 10.1111/age.13077
Pirosanto Y, Laseca N, Valera M, Molina A, Moreno-Millu00e1n M, Bugno-Poniewierska M, Ross P, Azor P, Demyda-Peyru00e1s S.Chromosomal abnormalities are a common cause of infertility in horses. However, they are difficult to detect using automated methods. Here, we propose a simple methodology based on single nucleotide polymorphism (SNP)-array data that allows us to detect the main chromosomal abnormalities in horses in a single procedure. As proof of concept, we were able to detect chromosomal abnormalities in 33 out of 268 individuals, including monosomies, chimerisms, and male and female sex-reversions, by analyzing the raw signal intensity produced by an SNP array-based genotyping platform. We also demonstrat...
Noscapine Acts as a Protease Inhibitor of In Vitro Elastase-Induced Collagen Deposition in Equine Endometrium.
International journal of molecular sciences    May 19, 2021   Volume 22, Issue 10 5333 doi: 10.3390/ijms22105333
Amaral A, Fernandes C, Szu00f3stek-Mioduchowska A, Rebordu00e3o MR, Skarzynski DJ, Ferreira-Dias G.Endometrosis is a reproductive pathology that is responsible for mare infertility. Our recent studies have focused on the involvement of neutrophil extracellular traps enzymes, such as elastase (ELA), in the development of equine endometrosis. Noscapine (NOSC) is an alkaloid derived from poppy opium with anticough, antistroke, anticancer, and antifibrotic properties. The present work investigates the putative inhibitory in vitro effect of NOSC on collagen type I alpha 2 chain () mRNA and COL1 protein relative abundance induced by ELA in endometrial explants of mares in the follicular or mid-lu...
Horse Clinical Cytogenetics: Recurrent Themes and Novel Findings.
Animals : an open access journal from MDPI    March 16, 2021   Volume 11, Issue 3 831 doi: 10.3390/ani11030831
Bugno-Poniewierska M, Raudsepp T.Clinical cytogenetic studies in horses have been ongoing for over half a century and clearly demonstrate that chromosomal disorders are among the most common non-infectious causes of decreased fertility, infertility, and congenital defects. Large-scale cytogenetic surveys show that almost 30% of horses with reproductive or developmental problems have chromosome aberrations, whereas abnormal karyotypes are found in only 2-5% of the general population. Among the many chromosome abnormalities reported in the horse, most are unique or rare. However, all surveys agree that there are two recurrent c...
Antioxidants in assisted reproductive technologies: An overview on dog, cat, and horse.
Journal of advanced veterinary and animal research    March 11, 2021   Volume 8, Issue 1 173-184 doi: 10.5455/javar.2021.h500
Ciani F, Maruccio L, Cocchia N, d'Angelo D, Carotenuto D, Avallone L, Namagerdi AA, Tafuri S.Assisted reproductive technologies (ARTs) are widely used as a tool to improve reproductive performance in both humans and animals. In particular, in the veterinary field, ARTs are used to improve animal genetics, recover endangered animals, and produce offspring in the event of subfertility or infertility in males or females. However, the use of ARTs did not improve the fertilization rate in some animals due to various factors such as the difficulty in reproducing an anatomical and humoral substrate typical of the natural condition or due to the increase in catabolites and their difficult eli...
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