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Topic:Fertility

Fertility in horses encompasses the physiological processes and factors influencing reproductive success in equine species. It involves the study of reproductive anatomy, endocrinology, and behavior, as well as the management practices that affect breeding outcomes. Key aspects include the estrous cycle, conception rates, and factors impacting stallion and mare fertility. Reproductive technologies such as artificial insemination, embryo transfer, and hormonal therapies are also explored to enhance breeding efficiency. This page compiles peer-reviewed research studies and scholarly articles that examine the biological mechanisms, management strategies, and technological advancements related to equine fertility.
Effects of holding and the addition of naloxone on vitrification of equine immature oocytes.
Theriogenology    February 28, 2025   Volume 239 117359 doi: 10.1016/j.theriogenology.2025.02.025
Gugole PM, Zannoni A, Forni M, Iacono E, Zambelli F, Merlo B.This study investigates the effects of overnight holding and naloxone (Nx) supplementation on the vitrification outcomes of equine immature oocytes. Oocytes were divided into six experimental groups based on treatment combinations: fresh (F) and held (H) control oocytes, oocytes vitrified with or without Nx (10 M) (VIT and VIT-Nx), oocytes vitrified after overnight holding with or without Nx (10 M) (H-VIT and H-VIT-Nx). They were assessed for survival, meiotic competence, intracellular oxidative stress, mitochondrial activity and distribution, apoptosis, and apoptotic gene expression. At sur...
Comparison of pregnant mare serum gonadotropin products with surprising differences in protein content.
Scientific reports    February 25, 2025   Volume 15, Issue 1 6824 doi: 10.1038/s41598-025-90833-3
Lösle M, Lin CW, Beil-Wagner J, Aebi M, Buch T.Equine chorionic gonadotropin (eCG) is a widely used hormone that synchronizes the female cycle and induces estrus in livestock. eCG is a heterodimeric glycoprotein composed of non-covalently linked α- and β-chains whose glycosylation profiles determine the in vivo activity of the hormone. The commercially available eCG products are crudely purified from the serum of pregnant mares, hence called pregnant mare serum gonadotropin (PMSG). Appropriate glycosylation of the protein is crucial for the correct binding to the receptor, receptor activation, and its half-life. The exact protein composi...
Enhanced Production and Functional Characterization of Recombinant Equine Chorionic Gonadotropin (rec-eCG) in CHO-DG44 Cells.
Biomolecules    February 14, 2025   Volume 15, Issue 2 289 doi: 10.3390/biom15020289
Byambaragchaa M, Park SH, Park MH, Kang MH, Min KS.Equine chorionic gonadotropin (eCG) hormone, comprising highly glycosylated α- and β-subunits, elicits responses similar to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in non-equid species. This study aimed to establish a mass production of recombinant eCG (rec-eCG) using CHO DG44 cells. Single-chain rec-eCG β/α was expressed in CHO DG44 cells. FSH- and LH-like activities were evaluated in CHO-K1 and HEK 293 cells expressing the equine LH/CG receptor (eLH/CGR), rat LH/CGR (rLH/CGR), and rFSHR. pERK1/2 activation and β-arrestin 2 recruitment were assessed in PathHunter ...
Strategic approaches to improve equine breeding and stud farm outcomes.
Veterinary world    February 13, 2025   Volume 18, Issue 2 311-328 doi: 10.14202/vetworld.2025.311-328
Khan IU, Khairullah AR, Khan AY, Rehman AU, Mustofa I.This review explores advanced strategies for enhancing fertility and optimizing reproductive outcomes in equine breeding programs. Horses, being seasonal breeders, present unique reproductive challenges influenced by environmental and physiological factors such as photoperiods, hormone cycles, and aging. Key approaches discussed include hormonal therapies, artificial light manipulation, and nutritional supplementation to improve ovulation and conception rates during the breeding season. Specific hormones such as gonadotropin-releasing hormone analogs, equine follicle-stimulating hormone, and p...
Transcriptome Analysis Reveals Equine Endometrium’s Gene Expression Profile Around Embryo Fixation.
Genes    February 1, 2025   Volume 16, Issue 2 doi: 10.3390/genes16020181
Ulaangerel T, Mu S, Sodyelalt J, Yi M, Zhao B, Hao A, Wen X, Han B, Bou G.The success or failure of embryo fixation is crucial for embryo attachment and later development. As an epithelial chorioallantoic placenta-type animal, the horse has a special process of embryo implantation, and the mechanism of embryo fixation in horses is still unclear. Methods: In this study, the structural and transcriptomic characteristics of endometrial tissue from the fixed and nonfixed sides of 20-day gestation embryos in Mongolian horses were investigated to search for important genes and potential molecular markers associated with the fixation phase of equine embryos. Results: A com...
Laparoscopic Inguinal Hernioplasty with a Polyether Ether Ketone Anchoring Device in Intact Male Horses Does Not Compromise Testicular Perfusion, Sperm Production or Motility Characteristics.
Animals : an open access journal from MDPI    January 31, 2025   Volume 15, Issue 3 doi: 10.3390/ani15030402
Vitoria A, Barrachina L, Romero A, Fuente S, de Blas I, Gil L, Vázquez FJ.A new surgical technique using a polyether ether ketone (PEEK) anchoring device for testicle-sparing laparoscopic inguinal hernioplasty in stallions was described in 2023 and is known as the PEEK harpoon technique (PHT). In breeding stallions, it is essential that the surgery is effective in preventing inguinal hernia but also that it does not impair the testicular function. This study aims to evaluate whether the PHT may affect testicular function. To achieve that, changes in the testicular blood flow, sperm production and motility characteristics were assessed 28 days after use of the PHT. S...
Sperm vitrification in horses and donkeys.
Journal of equine veterinary science    January 9, 2025   Volume 145 105340 doi: 10.1016/j.jevs.2024.105340
Hidalgo M.Sperm vitrification is an alternative freezing method, which includes high cooling rates and non-permeable cryoprotectants agents. The first attempt in equids was using the spheres technique by directly dropping small volumes of the sperm into liquid nitrogen. Later, vitrification was developed using 0.25 mL straws with outer covers, which resulted in similar progressive motility when compared to conventional freezing in donkeys (44.3 ± 15.0 % vs. 44.7 ± 18.2 %) or even higher in horses (48.2 ± 2.3 % vs. 37.3 ± 2.2 %). Subsequently, the vitrification of larger volumes of sperm in 0.5 mL st...
Ovarian steroids modulate mRNA expression of ECM associated genes and collagen deposition induced by TGF β1 in equine endometrium in vitro.
Scientific reports    January 2, 2025   Volume 15, Issue 1 538 doi: 10.1038/s41598-024-84250-1
Ana A, Agnieszka S, Marta CP, Pawel K, Dariusz S, Graça FD, Szóstek-Mioduchowska A.Equine endometrosis is a major cause of infertility in mares and is characterized by degenerative, functional and fibrotic changes in the endometrium with increased collagen (COL) deposition. Transforming growth factor (TGF)-β1 is one of the major pro-fibrotic factors involved in the excessive deposition of extracellular matrix (ECM) components in the equine endometrium. It has been demonstrated that ovarian steroids, specifically 17β-estradiol (E2) and progesterone (P4), not only regulate the cyclicity of the estrous cycle, but also have been implicated as anti- or pro-fibrotic factors. Thi...
Effects of Porcine Zona Pellucida Immunocontraception on Mare Body Condition and Foaling Season Length in Two Western Wild Horse Populations.
Animals : an open access journal from MDPI    December 9, 2024   Volume 14, Issue 23 doi: 10.3390/ani14233550
Rutberg AT, Grams KA.Wildlife managers and the public have expressed considerable interest in the use of contraception to help manage the populations of wild horses and burros (Equus caballus and E. asinus). Field testing has shown that two preparations of the porcine zona pellucida (PZP) vaccine, a simple emulsion (ZonaStat-H) and PZP-22 (which supplements ZonaStat-H with a controlled-release component) effectively prevent pregnancy in individual mares and can substantially reduce population foaling rates. To determine whether some PZP preparations might have secondary effects that harm treated mares or their foa...
The use of sex-sorted semen in horses.
Journal of equine veterinary science    December 5, 2024   Volume 145 105251 doi: 10.1016/j.jevs.2024.105251
Samper JC, Hernandez Aviles JC, Ramirez-Agamez LF, Love CC, Gonzalez-Marin C, Fleury P, Dini P, De La Fuente A, Foss R, Campos FL, Ross PJ.The application of sex-sorted semen in horses has historically lagged the bovine industry due to differences in the reproductive physiology and grater variability in equine semen quality. Recent advancements, including SuperGen™ sorters and Ultraplus™ semen processing methods developed by STgenetics® (Navasota, Texas), have significantly improved the efficiency of the sex-sorting process and reduced the sperm damage previously reported. To facilitate the broader use sex-sorted semen in the equine industry, it is essential to address challenges such as shipping cooled semen to a central so...
Fluctuations of antimüllerian hormone, ovarian follicular reserve, and antioxidant status throughout the estrous cycle in aged mares.
Open veterinary journal    November 30, 2024   Volume 14, Issue 11 3132-3143 doi: 10.5455/OVJ.2024.v14.i11.44
Aboelmaaty AM, Elgharieb AEA, El-Debaky HA, Alkhadrawy JMH, Abou-Ahmed MM, Ghallab AM.Senility influences fertility in women and companion animals, especially horses. Unassigned: This study aimed to investigate the effect of aging in horses on the daily changes in the dominant follicle (DF) dynamics and hemodynamics, antimüllerian hormone (AMH), enzymes, antioxidants, and ovarian hormones during the estrous cycle. Unassigned: Ovaries of old mares ( = 5, age >20 years) and young native mares ( = 6, age <10 years) were scanned during 6 different estrous cycles from March 2022 to August 2023 with Doppler ultrasound. The DF diameter and color area, the corpus luteum (CL) dia...
Improved embryo recovery through increased flushing media volume and the impact on unintended pregnancies in a single subject donor mare.
Journal of equine veterinary science    November 29, 2024   105246 doi: 10.1016/j.jevs.2024.105246
Sala-Ayala L, Cuervo-Arango J, Martinez-Bovi R.The case report discusses the implications of using different volumes of flushing media during embryo flushing (EF) procedures in a single donor mare that experienced multiple unintended pregnancies following negative EF outcomes. The study involved two sets of EF experiments over two years on a non-lactating Spanish purebred mare. The initial experiments (group 1, n=6) used 1L of flushing medium (Ringer´s Lactate), and subsequent tests (group 2, n=5) increased the volume to 2L, to assess whether a larger volume could enhance embryo recovery rates. After every EF procedure, no PGF2α was admi...
Oocyte holding and in vitro maturation duration between 28 and 34 hours do not affect equine OPU-ICSI outcomes.
Theriogenology    November 22, 2024   Volume 233 64-69 doi: 10.1016/j.theriogenology.2024.11.019
Broothaers K, Pascottini OB, Hedia M, Angel-Velez D, De Coster T, Peere S, Polfliet E, Van den Branden E, Govaere J, Van Soom A, Smits K.Previous studies in the horse highlight the potential benefit of prolonged in vitro maturation (IVM) (34 h) compared to short IVM (24 h) with or without prior oocyte holding, but little is known about the optimal IVM duration within this interval. To determine the effect of oocyte holding and duration of IVM ranged between 28 and 34 h on nuclear maturation, cleavage, blastocyst formation, and pregnancy rates, a retrospective study was performed in an equine clinical OPU-ICSI setting. The study included data of 2114 aspirated oocytes from 201 OPU-ICSI sessions. Duration of IVM was divided in...
Optimization of vitrification methods for equine oocytes.
Tissue & cell    November 19, 2024   Volume 91 102632 doi: 10.1016/j.tice.2024.102632
Du M, Li X, Bayinnamula , Wang N, Liu Y, Zhang L, Zhao Y, Dugarjaviin M.An important method for preserving equine germplasm is the cryopreservation of equine oocytes. Due to its ease, rapidity and affordability, vitrification freezing has taken over as the primary method of horse oocyte cryopreservation. The vitrification cryoprotectants utilized in this investigation were Ethylene glycol (E), Dimethyl sulfoxide (D), Sucrose (S), and Ficoll (F). According to the oocyte volume alteration, the treatment time was 39 s in equilibrium solution ED10 (10 % EG + 10 % DMSO), 32 s in equilibrium solution ED15 (15 % EG + 15 % DMSO), while 20 s in equilibrium so...
Warmblood fragile foal syndrome: Pregnancy loss in Warmblood mares.
Equine veterinary journal    November 14, 2024   Volume 57, Issue 4 915-923 doi: 10.1111/evj.14435
Kehlbeck A, Blanco M, Venner M, Freise F, Gunreben B, Sieme H.Fragile foal syndrome (FFS) or warmblood FFS Type 1 (WFFS) is a recessive, autosomal, hereditary, genetic defect causing late abortions, stillbirths and non-viable foals. Whether early pregnancy losses occur is unknown. Objective: To investigate how WFFS affects pregnancies and whether early pregnancy losses occur in WFFS matings and if there is a difference in pregnancy success between matings where both parents were allele carriers and those where only mare or stallion were WFFS carriers. Methods: Retrospective cohort study. Methods: Breeding records from a Warmblood stud farm were evaluated...
Analysis of stallion spermatozoa metabolism using Agilent Seahorse XFp Technology.
Animal reproduction science    November 3, 2024   Volume 271 107633 doi: 10.1016/j.anireprosci.2024.107633
Ortiz-Rodriguez JM, Bucci D, Tovar-Pascual L, Granata S, Spinaci M, Nesci S.Sperm metabolism consists of a sophisticated network of biochemical reactions and varies between species, resulting in different metabolic strategies for ATP production to maintain sperm functionality. ATP can be produced through glycolysis or in the mitochondria by oxidative phosphorylation (OXPHOS). Since OXPHOS is the predominant metabolic pathway in horses spermatozoa, various assessments of mitochondrial activity are used to evaluate fertility, utilizing techniques such as fluorescent probes analysed via microscopy or flow cytometry, and polarographic electrode assays to measure current f...
IVF with frozen-thawed sperm after prolonged capacitation yields comparable results to ICSI in horses: A morphokinetics study.
Theriogenology    November 1, 2024   Volume 232 39-45 doi: 10.1016/j.theriogenology.2024.10.032
Martin-Pelaez S, Fuente A, Takahashi K, Perez IT, Orozco J, Okada CTC, Neto CR, Meyers S, Dini P.Intracytoplasmic sperm injection (ICSI) is the current clinical practice for the in vitro production of equine embryos. The use of conventional fertilization methods such as in vitro fertilization (IVF), has historically been associated with poor success in horses. However, recent improvements have led to better outcomes with IVF, though only when using fresh semen, which limits its use in clinical practice. IVF remains in its infancy in equine reproduction, and several unknowns remain about the technique. One significant gap in knowledge concerns the morphokinetics of IVF embryos and how they...
Periovulatory anticoagulant therapy enhances embryo recovery rates in superovulated mares.
Theriogenology    October 22, 2024   Volume 231 182-189 doi: 10.1016/j.theriogenology.2024.10.018
Rodrigues LT, Segabinazzi L, Frasson M, Dell'Aqua C, Carmo MT, Takahira RK, Alvarenga M, Papa F.Although protocols for superovulation have been described in horses, this technique has been discouraged due to the low embryo recovery rates in superovulated mares. The reason for these poor results is poorly understood, but the formation of a blood clot in the ovulation fossa following ovulations has been hypothesized. Therefore, this study aimed to assess the safety and effect of periovulatory anticoagulant therapy on embryo recovery of superovulated mares. In experiment 1, five mares were assigned to receive five anticoagulant treatments in a crossover design: intravenous injections of 150...
A new hormonal protocol supports early development of in vitro-produced embryos after transfer to anoestrus mares.
Reproduction in domestic animals = Zuchthygiene    October 14, 2024   Volume 59 Suppl 3 e14622 doi: 10.1111/rda.14622
Muñoz-García CC, Luis-Calero M, Fernández-Hernández P, Pizarro-Hernández A, Fernández-González R, Gutiérrez-Adán A, Sánchez-Calabuig MJ....The present study aimed to evaluate whether primed anoestrus mares are suitable recipients for embryos produced by intracytoplasmic sperm injection (ICSI). Anoestrus was confirmed in four mares and daily doses of oestradiol benzoate (6 mg in total) over 5 days were administered; after 3 days of rest, oral altrenogest was administered at 0.088 mg/kg and embryos (1 to 5 embryos per mare; 15 in total) were transferred 3.5 days after progesterone onset. Uterine lavage was conducted 48 h after transfer. The results revealed an 80% embryo recovery rate, and among the retrieved embryos, 6...
Prolonged incubation of frozen-thawed equine spermatozoa for in vitro fertilization: A preliminary study using low temperature and INRA96 medium.
Reproduction in domestic animals = Zuchthygiene    October 14, 2024   Volume 59 Suppl 3 e14593 doi: 10.1111/rda.14593
Luis-Calero M, Muñoz-García CC, Fernández-Hernández P, Macías-García B, González-Fernández L.A protocol for conventional in vitro fertilization (IVF) in horses using fresh semen has been described, using a prolonged incubation in FERT-TALP medium (22 h) at 38.2°C in the presence of penicillamine, hypotaurine and epinephrine (PHE). Our work aimed to develop a protocol that maintains quality parameters in frozen-thawed equine spermatozoa incubated for 22 h in the presence of PHE using different media (FERT-TALP and INRA96) and incubation temperatures (30 and 38.2°C). Twelve frozen ejaculates from four stallions were thawed and then incubated in either FERT-TALP or INRA96 with PHE...
Effect of refreezing extender on stallion sperm quality and embryo production after intracytoplasmic sperm injection.
Theriogenology    October 9, 2024   Volume 231 29-35 doi: 10.1016/j.theriogenology.2024.10.006
Brito LFC, Felix MR, Linardi RL, Martinez de Andino EV, Balamurugan NS, Hernández-Avilés C, Hinrichs K.Intracytoplasmic sperm injection (ICSI) is a valuable assisted reproduction technology in clinical practice, especially when semen availability is limited. Since the number of sperm required per ICSI cycle is much less than the number of sperm available in a standard straw of frozen semen, refreezing semen at lower sperm concentrations could yield multiple straws for ICSI use. However, there is little data on the effect of sperm refreezing on ICSI outcomes, especially on the effect of extender used for refreezing. The objective of the present study was to evaluate the effect of refreezing exte...
RNA Sequencing of Sperm from Healthy Cattle and Horses Reveals the Presence of a Large Bacterial Population.
Current issues in molecular biology    September 19, 2024   Volume 46, Issue 9 10430-10443 doi: 10.3390/cimb46090620
Navarrete-López P, Asselstine V, Maroto M, Lombó M, Cánovas Á, Gutiérrez-Adán A.RNA molecules within ejaculated sperm can be characterized through whole-transcriptome sequencing, enabling the identification of pivotal transcripts that may influence reproductive success. However, the profiling of sperm transcriptomes through next-generation sequencing has several limitations impairing the identification of functional transcripts. In this study, we explored the nature of the RNA sequences present in the sperm transcriptome of two livestock species, cattle and horses, using RNA sequencing (RNA-seq) technology. Through processing of transcriptomic data derived from bovine and...
Effect of protein acetylation on capacitation of stallion sperm.
Journal of equine veterinary science    September 18, 2024   Volume 142 105195 doi: 10.1016/j.jevs.2024.105195
Aguiar LH, Pinto CRF.Sperm capacitation is considered the main factor limiting conventional in vitro fertilization (IVF) in horses. A recent scientific breakthrough in sperm processing for IVF in horses has resulted in embryos and foals being produced; however, various aspects of the IVF process remain to be fully elucidated. Lysine acetylation has been shown to play a role in sperm capacitation in several species and the objective of this study was to detect and evaluate this process in the horse. Ejaculates of two stallions were collected and incubated in different conditions with deacetylase inhibitors to induc...
Maturational competence of equine oocytes is associated with alterations in their ‘cumulome’.
Molecular human reproduction    September 17, 2024   Volume 30, Issue 9 doi: 10.1093/molehr/gaae033
No abstract available
Equine ART and antral follicle count: Can we deepen our understanding to improve outcomes?
Reproduction in domestic animals = Zuchthygiene    September 5, 2024   Volume 59 Suppl 2 e14625 doi: 10.1111/rda.14625
Le Breton A, Lewis N.Assisted reproductive technologies (ARTs) are performed worldwide in the equine industry to produce genetically valuable foals. Among them, ovum pick up (OPU) combined with intra-cytoplasmic sperm injection (ICSI) can now be more efficient than embryo transfer (ET) under optimal conditions. However, OPU is not a benign procedure for the mare and the process is costly. Improved efficiency is therefore in the interest of everyone, maximizing mare welfare and optimizing economics for the client. One of the key factors of success is the antral follicle count (AFC) at the time of OPU and subsequent...
The Use of Genomic Screening for the Detection of Chromosomal Abnormalities in the Domestic Horse: Five New Cases of 65,XXY Syndrome in the Pura Raza Español Breed.
Animals : an open access journal from MDPI    September 3, 2024   Volume 14, Issue 17 2560 doi: 10.3390/ani14172560
Valera M, Karlau A, Anaya G, Bugno-Poniewierska M, Molina A, Encina A, Azor PJ, Demyda-Peyrás S.Sex chromosomal abnormalities are a well-established cause of reproductive failure in domestic horses. Because of its difficult diagnosis, the Pura Raza Español breeding program established a routine screening for chromosomal abnormalities in all the horses prior to enrolling in the studbook. This genomic procedure combines an initial assessment based on the results from Short Tandem Repeat (STR) parentage testing followed by a Single-Nucleotide Polymorphism (SNP) based copy number aberration (CNA) confirmative analysis in positive cases. Using this methodology, we identified five new individ...
Cervix-Deep Rectal Temperature Differential on the Day of Ovulation is Correlated With Embryo Recovery Results in Mares.
Reproduction in domestic animals = Zuchthygiene    August 31, 2024   Volume 59, Issue 9 e14716 doi: 10.1111/rda.14716
Funes J, Ismer A, Hoogewijs M, Wilsher S.Variations in temperature throughout the reproductive tract have been noted in many species. A recent study found the cervix-rectum temperature differential (CR-TD) in cattle was related to fertility. The present study aimed to assess the CR-TD in mares around the time of ovulation and relate it to embryo recover. Over 52 cycles, mares were inseminated with a fertile stallion and embryo recovery was undertaken on Day 7 post ovulation. Further 10 control mares were not inseminated. Rectal and cervical temperatures were measured using a precision thermometer on four or five occasions: the day of...
Reproductive Disorders in Donkeys: Current Evidence and Update.
Animals : an open access journal from MDPI    August 28, 2024   Volume 14, Issue 17 2494 doi: 10.3390/ani14172494
Wang Z, Zeng S, Wang Y, Wang T, Qu H, Zhu Y, Li J.Reproductive disorders in donkeys present a significant challenge to their health and welfare, impacting their roles in agriculture, conservation, and companionship. With the development of large-scale donkey farming in recent years, reproductive disorders have become a limiting factor for the expansion of the donkey population. In general, donkeys suffer from a similar array of diseases like horses, but little is known about the specificities of donkey reproductive disorders. This review synthesizes current knowledge on the pathogenesis, distribution, presentation, diagnosis, treatment, and p...
In vitro, the aging of stallion spermatozoa at 22 °C is linked to alteration in Ca2+ and redox homeostasis and may be slowed by regulating metabolism.
Theriogenology    August 22, 2024   Volume 229 127-137 doi: 10.1016/j.theriogenology.2024.08.021
Becerro-Rey L, Martín-Cano FE, Fabres Robaina Sancler-Silva Y, Gil MC, Ortega-Ferrusola C, Aparicio IM, Gaitskell-Phillips G, da Silva-Álvarez E....Conservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24-48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present i...
Molecular detection of Chlamydia abortus in endometrial biopsies of mares from western Canada. Ricard RM, Wobeser B. is a reported cause of infertility and endometritis in sheep, cattle, and pigs; however, the association between uterine disease and is poorly understood in horses. Recently, a high prevalence of in equine aborted chorioallantoises was reported in horses in western Canada. Based on this high prevalence, investigation into the effects of on infertility and endometritis in western Canadian mares is prudent. We examined 98 formalin-fixed, paraffin-embedded endometrial biopsies from western Canada submitted between 2014 and 2022 using a -specific 16S rRNA PCR test; 40 samples tested positive f...