Reproductive technology in horses encompasses a range of scientific techniques and procedures aimed at assisting and enhancing equine reproduction. These technologies include artificial insemination, embryo transfer, and cryopreservation of gametes and embryos. They are employed to improve breeding efficiency, manage genetic diversity, and preserve valuable genetic material. Artificial insemination involves the collection and introduction of semen into the mare's reproductive tract, while embryo transfer allows for the harvesting and implantation of embryos from donor to recipient mares. Cryopreservation involves freezing and storing sperm, oocytes, or embryos for future use. This page compiles peer-reviewed research studies and scholarly articles that investigate the methodologies, applications, and outcomes of reproductive technologies in equine breeding and management.
da Silva-Álvarez E, Gómez-Arrones V, Correa-Fiz F, Martín-Cano FE, Gaitskell-Phillips G, Carrasco JJ, Rey J, Aparicio IM, Peña FJ, Alonso JM....This study aims to unveil potential differences in the vaginal and uterine microbiomes in mares and jennies, and to identify possible mechanisms involved in uterine immune homeostasis. The microbiota was characterized using 16S rRNA sequencing, and the uterine proteome was analyzed using UHPLC/MS/MS in 18 samples from healthy mares and 14 from jennies. While taxonomic analysis revealed high interspecies similarities, β-diversity analysis showed distinct clustering, with only two vaginal taxa and five uterine taxa differing between species. Despite compositional differences, PICRUSt analysis s...
Sinovich M, Monné Rodriguez J, Pieńkowska-Schelling A, Schelling C, Kelly PG.Introduction Monorchidism is a rarely described condition in the horse and is not to be confused with cryptorchidism. The diagnosis is challenging and confirmed by surgery and histology in combination with hormonal assays. This report describes, to the best of the author's knowledge, the first case of monorchidism and abdominal cryptorchidism of the developed testicle in a horse. Methods An Irish Cob underwent laparoscopic castration for removal of bilateral cryptorchid testicles. At surgery the horse was diagnosed as a monorchid with the testicle retained intra-abdominally. Histopathological,...
Abah KO, Ligocka-Kowalczyk Z, Itodo JI, Ameh G, Partyka A, Nizanski W.Accurately predicting male fertility is crucial for the animal breeding industry due to its significant economic implications. Existing literature suggests that mammalian fertility is partially dependent on sperm DNA integrity. However, routine semen analysis often fails to detect DNA damage and does not consistently correlate with field fertility outcomes. While assessing sperm DNA integrity provides valuable biological insights, its role in diagnosing animal infertility remains uncertain. Results: This meta-analysis evaluated the association between sperm DNA fragmentation (SDF) and fertilit...
Muñoz-Jurado A, Requena F, Agüera EI, Escribano BM.Anti-Müllerian hormone (AMH) is a dimeric glycoprotein belonging to the superfamily of the transforming growth factor-β. Due to the discovery of AMH functions, relative to the ovarian function, it is being postulated as being a highly important marker in studies on mammalian reproduction. Therefore, the objective of this review was to describe the role of this hormone in different reproductive aspects of female mammals, taking women, cows, and mares as reference species. The relationship between ovarian reserve and AMH was analysed, and it has been verified that there is a relationship betwe...
Sultan T, Chaudhary AK, Mehta JS, Mehra R, Manuja A, Talluri TR, Bhattacharya TK.Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Different substances and compounds can be added to semen extenders to improve sperm quality. Objective: To investigate the effect of supplementing semen extender with zinc nanoparticles (ZnONPs) and gold nanoparticles (AuNPs) on cooled and frozen-thawed spermatozoa of Marwari stallion. Methods: A total of 20 ejaculates from four Marwari ...
Cuervo-Arango J, Sala-Ayala L, Márquez-Moya A, Martínez-Boví R.The objective of this study was to determine the effect of two aspiration pressures (75 vs. 150 mmHg), the follicle flushing method (injection pump controlled by a foot pedal vs. a plastic syringe) and the twisting of the OPU needle on oocyte recovery and in vitro embryo production. OPU data from a total of 104 warmblood sport mares belonging to a commercial OPU-ICSI program were collected as part of a prospective study split into three experiments. Each mare was used only once for OPU. In Experiment 1, the mares' follicles were aspirated using either a high aspiration pressure (flow rate of 1...
Felix MR, Dobbie T, Woodward E, Linardi R, Okada C, Santos R, Hinrichs K.We recently reported successful equine IVF using fresh semen pre-incubated for a prolonged period (22 h) before co-culture with oocytes. In this study, we evaluated the feasibility of equine IVF with frozen-thawed sperm and evaluated capacitation-related changes in these sperm over the pre-incubation period. Sperm selected via a commercial sperm separation device (SSD) yielded significantly higher fertilization than did sperm selected by swim-up or by colloid centrifugation. Using the SSD method, fertilization rates with sperm pre-incubated for 15 min, 3 h, 6 h, and 9 h were 7.1, 22.2, 38...
Cuervo-Arango J, Necchi D, Clutton-Brock A, Profaska M, Crabtree J, Claes A.This retrospective clinical study describes different techniques for transvaginal follicle aspiration in mares and compares results from 5 different commercial ovum pick-up (OPU) clinics in which the same operator aspirated mares using different systems and equipment: Clinic 1 (n = 42 mares, two-operator OPU technique, double-lumen-echogenic-tip needle, and manual syringe-assisted flushing of follicles), Clinic 2 (n = 28 mares, single-operator-OPU-technique, double-lumen-echogenic-tip needle, infusion pump controlled by foot-pedal for follicle flushing), Clinic 3 (n = 18 mares, sin...
Wang B, Duan W, Zhao J, Bai D.Once a mare experiences parturition abnormalities, the outcome between a live foal and a stillborn can change rapidly. Automated detection of mare parturition and timely human intervention is crucial to reducing risks during mare and foal parturition. This paper addresses the challenges of manual monitoring of parturition in large-scale equine facilities due to the unpredictability of mare parturition timing, proposing an algorithm for detecting mare parturition through a balanced multi-scale feature fusion based on an improved Libra RCNN. Initially, a ResNet101 backbone network incorporating ...
Hernández-Avilés C, Varner DD, Ramírez-Agámez L, Samper JC, Love CC.In the current study, we report the effect of different commercially available semen freezing extenders utilized for the "test-freeze" procedure for 13 years (2010-2023) as part of a commercial program of stallion sperm cryopreservation. Ejaculates obtained from sexually active and healthy stallions (n = 124) were cryopreserved using up to five commercially available semen freezing extenders (Lactose-EDTA [LE], MFR5, CryoMax LE [CMLE], CryoMax MFR5 [CMMFR5] or BotuCrio [BC]). Post-thaw sperm motility (total motility - TM [%]; progressive motility - PM [%]; and curvilinear velocity - VCL [μm...
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 ...
Perera TRW, de Ruijter-Villani M, Gibb Z, Nixon B, Sheridan A, Stout TAE, Swegen A, Skerrett-Byrne DA.Embryo-maternal signaling during the establishment of pregnancy in horses remains one of the biggest mysteries in large animal physiology. Early pregnancy loss represents a major source of economic loss to the breeding industry. This study aimed to investigate the systemic changes associated with early pregnancy by mapping the proteome of blood plasma at 14 days in pregnant and non-pregnant mares. Methods: Plasma proteomes were analysed in commercially bred pregnant (n = 17) and non-pregnant (n = 17) Thoroughbred mares at 14 days after ovulation, using high-resolution mass spectrometry. D...
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...
Arnold LC, Stout TAE, Beitsma MM, Deelen CC, Rizzo M, Leemans B, Galli C, Colleoni S, Lazzari G, Barandalla M, Claes ANJ.It is not known whether the likelihood of pregnancy after transfer of cryopreserved equine in vitro produced (IVP) blastocysts is related to post-thaw morphological characteristics, or could be impacted by other factors such as embryo biopsy prior to cryopreservation. This study examined associations between post-thaw morphological appearance of equine IVP embryos, and biopsy prior to slow-freezing, on the likelihood of pregnancy. Biopsy of IVP blastocysts was performed by harvesting cells herniated through the intra-cytoplasmic sperm injection (ICSI)-derived hole in the zona pellucida (ZP). M...
Azcona F, Molina A, Demyda-Peyrás S.Analyzing genetic variability and inbreeding trends is essential for effective breed management in animal populations. To this, the characterization of runs of homozygosity (ROH) provides a good genomic approach to study the phenomena. The Polo Argentino (PA) breed, globally recognized as the best adapted to playing polo, is known for its strong influence of Thoroughbreds, intense selective breeding, and extensive use of reproductive biotechnologies. This study investigates the PA's genomic variability, by characterizing the ROH landscape and identifying ROH islands (ROHi) as potential genomic...
Piotrowska-Tomala KK, Szóstek-Mioduchowska A, Jonczyk AW, Drzewiecka EM, Wrobel MH, Hojo T, Ferreira-Dias G, Skarzynski DJ.Endometrosis (chronic degenerative endometritis) results in morphological changes in the equine endometrium and impairs its secretory function. However, the effect of this condition on the myometrium remains unclear. Lysophosphatidic acid (LPA) may affect female reproductive function and embryo transport by influencing uterine contractility through its receptors (LPARs). The objective of this study was to determine myometrial LPAR1-6 mRNA transcription, and the effects of LPA on myometrial contractions in mares with endometrosis during the mid-luteal and follicular phases of the estrous cycle....
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...
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...
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...
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...
Cerveira-Pinto M, Wójtowicz A, Pires MA, Kordowitzki P, Skarzynski D, Ferreira-Dias G, Szóstek-Mioduchowska A, Amaral A.Increased synthesis and deposition of collagen (COL) in the extracellular matrix (ECM) of equine endometrium contributes to endometrosis. Toll-like receptors (TLRs) are transmembrane receptors involved in the innate immune response, recognized for their role in antigen recognition and previously associated with equine endometritis. The TLRs not only recognize pathogen-associated molecular patterns but also regulate inflammations, fibrosis and cancer. The aim of this study was to explore the relationship between TLR expression at different stages of Kenney and Doig's (K-D) grading and COL1 exp...
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...
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...
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...
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...
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...
Lindinger H, Wehrend A.Various systems are available for birth monitoring in horses, whereby a distinction must be made between methods for more accurate prediction of the date of birth in order to intensify monitoring of the mare in a timely manner as well as methods for detecting individuals that are in labor. Basically, it should be noted that there are almost no studies that compare different methods on the same population of mares. As the time of birth approaches, physiological parameters of mare and fetus change, but their variability is too high to predict the exact parturition time point prospectively. The b...
Elkhawagah AR, Donato GG, Poletto M, Martino NA, Vincenti L, Conti L, Necchi D, Nervo T.This study aimed to investigate the effect of mitochondria-targeted antioxidants (Mitoquinone, MitoQ) on the quality of frozen-thawed stallion semen. Semen samples collected from three fertile stallions aged 10 - 13 years, were filtered, centrifuged in a skimmed milk-based extender, and diluted to a final concentration of 50 × 106 sperm/mL in freezing medium. Diluted semen was divided into five experimental groups supplemented with MitoQ at concentrations of 0 (control), 25, 50, 100, and 200 nM and then subjected to freezing after cooling and equilibration. After thawing, semen was evaluated ...
Mahmood K, Hassan M, Channa AA, Ghafoor A, Riaz A.The present study aimed to evaluate and compare the overall and breed-specific seasonal breeding patterns, fertility rates, cyclicity, and follicular dynamics of Arab, Thoroughbred, and Percheron mares under the subtropical conditions of Pakistan. Methods: A retrospective analysis of climatic data and breeding records of eleven breeding studs spanning four years (2020-2023) was made to find out the overall seasonality in the breeding pattern of mares. Fifty mares of each breed (n = 150 in total) were scanned by ultrasonography for a calendar year to find the cyclicity pattern and follicular ...
Flores JG, Bueno VC, Bastos HBA, Rechsteiner SMDEF.Reproductive biotechniques in the equine species have advanced in the last decade and horse breeders have started to question the possibilities of interfering in the determination of foal sex. The aim of the present study was to verify whether the variables mares and stallion's age, side of the ovary containing the preovulatory follicle, preovulatory follicle diameter, time between breeding and ovulation, and ovulation inducing hormones influence the sex of the foal. A total of 259 reproductive cycles of 160 mares and 22 Thoroughbred stallions were used. Statistical analysis was performed usin...
Klohonatz KM, Hess AM, Hansen TR, Squires EL, Bouma GJ, Bruemmer JE.The mechanism for maternal recognition of pregnancy (MRP) in horses is unknown. To maintain a pregnancy, a mobile conceptus must be recognized by the uterus before d 14 postovulation (PO). This recognition prevents endometrial secretion of PGF2α on d14 through 16, which would otherwise initiate luteolysis. The objective of this study was to evaluate gene expression in the endometrium of pregnant and nonpregnant mares during and after MRP to identify possible genes involved during this time. Twelve normally cycling mares were used in a crossover design and randomly assigned to a specific colle...
Peña FJ, Plaza Davila M, Ball BA, Squires EL, Martin Muñoz P, Ortega Ferrusola C, Balao da Silva C.The traditional assessment of stallion sperm comprises evaluation of sperm motility and membrane integrity and identification of abnormal morphology of the spermatozoa. More recently, the progressive introduction of flow cytometry is increasing the number of tests available. However, compared with other sperm structures and functions, the evaluation of mitochondria has received less attention in stallion andrology. Recent research indicates that sperm mitochondria are key structures in sperm function suffering major changes during biotechnological procedures such as cryopreservation. In this p...
Bazer FW, Vallet JL, Roberts RM, Sharp DC, Thatcher WW.Conceptuses produce steroids, prostaglandins, proteins and possibly other unidentified agents which may play a role in the establishment and maintenance of pregnancy. A key event in this process is protection of the corpus luteum (CL) from the luteolytic activity of prostaglandin (PG) F-2 alpha of uterine origin. Oestrogens produced by the pig conceptuses between Days 11 and 16 appear to exert an antiluteolytic effect resulting in the sequestering of PGF-2 alpha within the uterine lumen. Failure of the pregnant uterus to release PGF-2 alpha in an endocrine fashion, therefore, allows for mainte...
Alvarenga MA, Papa FO, Ramires Neto C.The use of stallion frozen semen minimizes the spread of disease, eliminates geographic barriers, and preserves the genetic material of the animal for an unlimited time. Significant progress on the frozen thawed stallion semen process and consequently fertility has been achieved over the last decade. These improvements not only increased fertility rates but also allowed cryopreservation of semen from "poor freezers." This article reviews traditional steps and new strategies for stallion semen handling and processing that are performed to overcome the deleterious effects of semen preservation a...
Kareskoski M, Katila T.Seminal plasma is a mixture of secretions produced in the testes, epididymides and accessory sex glands, and ejaculated as several consecutive fluid fractions. The composition of seminal plasma and the effects on sperm longevity vary between fractions and individual stallions. This review focuses on the sequence of ejaculation, constituents of seminal plasma and their potential use as fertility markers as well as the influence of seminal plasma on spermatozoa during storage.
Alves KA, Alves BG, Gastal GD, de Tarso SG, Gastal MO, Figueiredo JR, Gambarini ML, Gastal EL.Ovarian tissue collected by biopsy procedures allows the performance of many studies with clinical applications in the field of female fertility preservation. The aim of the present study was to investigate the influence of reproductive phase (anestrous vs. diestrous) and ovarian structures (antral follicles and corpus luteum) on the quality, class distribution, number, and density of preantral follicles, and stromal cell density. Ovarian fragments were harvested by biopsy pick-up procedures from mares and submitted to histological analysis. The mean preantral follicle and ovarian stromal cell...
Caillaud M, Duchamp G, Gérard N.A growing body of evidence suggests that the interleukin-1 system is involved in periovulatory events. Previous work from our lab demonstrated that in the mare, interleukin-1beta (IL-1beta) increases the ovulatory rate of metaphase II oocytes. The present study was conducted to analyze in vivo the effect of IL-1 on oocyte cytoplasmic maturation, ovulation and pregnancy rate. In the present work, IL-1beta (experiment 1, n = 13; experiment 2, n = 25) and interleukin-1RA (IL-1RA; experiment 1, n = 25) were injected intrafollicularly by using the transvaginal ultrasound-guided injection method. In...
Claes A, Stout TAE.In vitro embryo production (IVEP) via Ovum Pick-Up (OPU) and Intracytoplasmic Sperm Injection (ICSI) has become a popular breeding technique in Warmblood mares because of the high success rate and several practical advantages. IVEP offers a solution for a variety of reproductive issues including, but not limited to, sub-fertility in stallions or mares, poor quality or scarce frozen semen, difficulty in synchronizing donor and recipient mares, and inefficient use of recipient mares. In 515 OPU-ICSI sessions performed in our facility in 2021, a mean of 25.9 antral follicles were aspirated yield...
Jacobson CC, Choi YH, Hayden SS, Hinrichs K.Oocytes may be collected from live mares from either the stimulated preovulatory follicle or from all visible immature follicles. We evaluated the yield of mature oocytes, and of blastocysts after intracytoplasmic sperm injection (ICSI), for both follicle types. In Experiment 1, mares were assigned to Progesterone (1.2g biorelease progesterone weekly) or Control treatments. Transvaginal aspiration of all follicles was performed every 14 d. Overall, 596 follicles were aspirated, with a 54% oocyte recovery rate. There was no difference between treatments in number of follicles punctured (9.0 to ...
Miró J, Marín H, Catalán J, Papas M, Gacem S, Yeste M.In the donkey, artificial insemination (AI) with frozen-thawed semen is associated with low fertility rates, which could be partially augmented through adding seminal plasma (SP) and increasing sperm concentration. On the other hand, post-AI endometrial inflammation in the jenny is significantly higher than in the mare. While previous studies analyzed this response through recovering Polymorphonuclear Neutrophils (PMN) from uterine washings, successive lavages can detrimentally impact the endometrium, leading to fertility issues. For this reason, the first set of experiments in this work inten...
Hurtt AE, Landim-Alvarenga F, Seidel GE, Squires EL.Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol. Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EFS) for 30 sec and then to 5 M ethylene...
Sandra O, Charpigny G, Galio L, Hue I.In mammalian species, endometrial receptivity is driven by maternal factors independently of embryo signals. When pregnancy initiates, paracrine secretions of the preattachment embryo are essential both for maternal recognition and endometrium preparation for implantation and for coordinating development of embryonic and extraembryonic tissues of the conceptus. This review mainly focuses on domestic large animal species. We first illustrate the major steps of preattachment embryo development, including elongation in bovine, ovine, porcine, and equine species. We next highlight conceptus secret...
Oldenhof H, Wolkers WF, Sieme H.In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow cooling from room temperature to 5 °C, packaging of insemina...
Morrell JM, Johannisson A, Dalin AM, Rodriguez-Martinez H.The objective of the current study was to optimize the volumes of Androcoll-E and sperm sample used in various sizes of centrifuge tube to scale up single-layer centrifugation (SLC) for routine use in the field. Although sperm suspensions of equivalent quality were produced using Androcoll-E in small and large tubes, the sperm yield was much lower in the latter (P<0.001). In contrast, in 200-mL tubes (XL), the yields were approximately 25% higher than those for the small tubes. An increased volume (4.5 mL) of extended ejaculate in small tubes (SLC-Inc) or 15 to 18 mL extended ejaculate on 1...
Carnevale EM, Ramirez RJ, Squires EL, Alvarenga MA, Vanderwall DK, McCue PM.In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantl...
McDowell KJ, Sharp DC, Grubaugh W, Thatcher WW, Wilcox CJ.Cycling pony mares were bred and used to test the effect of restricted conceptus mobility on luteal maintenance (i.e. maternal recognition of pregnancy). In Experiment 1, uterine horns were ligated to restrict conceptus mobility to one uterine horn, Group 1; one horn plus the uterine body, Group 2; or one horn, the body and approximately 80% of the second horn, Group 3. Pregnancies were monitored with real-time ultrasonography. Four of five mares in Group 1 and two of four mares in Group 2 returned to estrus (Day 16.0 +/- 1.9 and 14.5 +/- 0.7, respectively) and subsequently lost the embryonic ...
Rose BV, Firth M, Morris B, Roach JM, Wathes DC, Verheyen KLP, de Mestre AM.Therapeutic practices in equine reproductive medicine have dramatically evolved over the last 20 years but current usage is not described. The aims of this study were to provide a description of medication use and clinical findings of reproductive examinations alongside measures of reproductive efficiency in thoroughbreds. A prospective cohort study was conducted in the 2013 and 2014 breeding seasons. Mare and stallion details, information on veterinary interventions and findings of reproductive ultrasound scans were collected using questionnaires and entered into a custom-designed Microsoft A...
Walter J, Huwiler F, Fortes C, Grossmann J, Roschitzki B, Hu J, Naegeli H, Laczko E, Bleul U.Maturation of oocytes under in vitro conditions (IVM) results in impaired developmental competence compared to oocytes matured in vivo. As oocytes are closely coupled to their cumulus complex, elucidating aberrations in cumulus metabolism in vitro is important to bridge the gap towards more physiological maturation conditions. The aim of this study was to analyze the equine "cumulome" in a novel combination of proteomic (nano-HPLC MS/MS) and metabolomic (UPLC-nanoESI-MS) profiling of single cumulus complexes of metaphase II oocytes matured either in vivo (n = 8) or in vitro (n = 7). Re...
Gil L, Olaciregui M, Luño V, Malo C, González N, Martínez F.In recent years, there has been an increased interest in new preservation techniques that facilitate sperm storage and distribution, with freeze-drying (FD) having been proposed as an alternative method for sperm preservation and maintenance of genetic resources in different animal species. FD is a method in which frozen material is dried by sublimation of ice, thereby involving a direct transition from a solid (ice) to a vapour (gas) phase. One of the main advantages of FD is that nitrogen and dry ice are no longer required for the storage and shipment of frozen sperm, which can be stored at ...
Dell'Aquila ME, Cho YS, Minoia P, Traina V, Lacalandra GM, Maritato F.The aim of this study was to compare the effect of the addition of follicular fluid (FF) collected from preovulatory follicles with that of oestrous mare serum (EMS) (acting as the control) to TCM-199 medium on the in-vitro maturation, fertilization and development of equine cumulus-enclosed oocytes. Oocytes (<30 mm in diameter) were obtained from the ovaries of slaughtered mares. After in-vitro maturation in the presence of the two supplements, their fertilization, cleavage and developmental potential were compared after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm inje...
Thompson RE, Meyers MA, Veeramachaneni DNR, Pukazhenthi BS, Hollinshead FK.Organoids are a type of three-dimensional (3D) cell culture that more closely mimic the in vivo environment and can be maintained in the long term. To date, oviductal organoids have only been reported in laboratory mice, women, and cattle. Equine oviductal organoids were generated and cultured for 42 days (including 3 passages and freeze-thawing at passage 1). Consistent with the reports in mouse and human oviductal organoids, the equine oviductal organoids revealed round cell clusters with a central lumen. Developing a 3D model of the mare oviduct may allow for an increased understanding of t...
Wilsher S, Clutton-Brock A, Allen WR.A total of 78 day 10 horse embryos were transferred non-surgically to recipient mares that had ovulated 9, 7, 6, 5, 4, 3, 2 or 1 day after (negative asynchrony), on the same day (synchronous), or 2 or 4 days before (positive asynchrony) the donor (n=6 or 8 mares per group). Pregnancy rates between 100% (6/6) and 63% (5/8) were seen in recipient mares that were between +2 and -6 days asynchronous. Embryo survival to the heartbeat stage declined in recipients that were -7 days asynchronous and no embryos survived in recipients that were -9 days asynchronous. Irrespective of uterine asynchrony, c...
Bucca S, Carli A, Buckley T, Dolci G, Fogarty U.The present study describes the effect of a single dose of dexamethasone administered to mares at time of breeding. In an initial experiment, the authors investigated safety of treatment. In a second experiment the effect of treatment on the uterine environment, fetal development and pregnancy outcome was examined. In the final part of the study, mares susceptible to persistent mating induced endometritis were identified, by means of a risk factor score system and the effect of treatment evaluated. Results indicated that dexamethasone administered at breeding time did not negatively impact on ...
Felix MR, Turner RM, Dobbie T, Hinrichs K.Methods for standard in vitro fertilization have been difficult to establish in the horse. We evaluated whether prolonged sperm pre-incubation would support subsequent fertilization. Fresh sperm were pre-incubated with penicillamine, hypotaurine, and epinephrine (PHE) for 22 h. Co-incubation of cumulus-oocyte complexes (COCs) for 6 h yielded 43% fertilization; culture of presumptive embryos yielded 21% blastocysts. Sperm incubated similarly, but without PHE, did not fertilize oocytes. Use of extended semen in the system yielded 54% blastocysts and was applied in subsequent experiments. Trans...
The Journal of heredityMay 1, 1984
Volume 75, Issue 3 220-224 doi: 10.1093/oxfordjournals.jhered.a109916
Cothran EG, MacCluer JW, Weitkamp LR, Pfennig DW, Boyce AJ.The relationship between inbreeding and both conception and foaling rates in Standardbred horses (trotters and pacers) was examined for 1194 breeding years. There was a statistically significant (P less than 0.05) trend for conception and foaling rates to decrease with increased inbreeding; however, this relationship accounted for less than 2 percent of the variation. Additionally, the relationship between reproductive performance and inbreeding was not consistent between trotters and pacers. For trotters (F = 0.103) there was a trend for an increase in conception and foaling rates with increa...
Macías García B, Morrell JM, Ortega-Ferrusola C, González-Fernández L, Tapia JA, Rodriguez-Martínez H, Peña FJ.The present study attempted to select the subpopulation of stallion spermatozoa that best survived a conventional freezing and thawing procedure, using centrifugation of post-thawed semen samples through a single layer of a glycidoxypropyltrimethoxysilane-coated silica colloid with a species-specific formulation (Androcoll-E). Sperm motility, sperm chromatin structure, membrane integrity and mitochondrial membrane potential were studied in filtered and non-filtered spermatozoa. Single-layer centrifugation (SLC) using Androcoll-E significantly improved all the sperm parameters studied, implying...
Tharasanit T, Colleoni S, Galli C, Colenbrander B, Stout TA.Vitrifying oocytes is a potentially valuable means of preserving the female germ line, but significantly compromises oocyte developmental competence. This study examined the hypothesis that the cumulus complex protects the oocyte during vitrification. Vitrified-warmed immature cumulus oocyte complexes (COCs) were labelled with a plasma membrane impermeant DNA marker (ethidium homodimer-1) to examine the percentage and location of dead cumulus cells, and to investigate the effect of the proportion of dead cells (+1,+2 or +3) on the success of in vitro maturation (IVM). Further, oocytes were lab...
Cuervo-Arango J, Claes AN, Stout TA.Advancing maternal age is known to negatively affect fertility in the horse. This age-related decrease in fertility has been linked primarily to reduced oocyte quality rather than to impaired uterine function. In the past decade, the use of ovum pick-up (OPU) and ICSI to produce foals has rapidly gaining popularity amongst sport horse breeders. However, it is not yet known how maternal age influences the efficiency of a commercial OPU-ICSI program and whether the age effect is similar to that observed for other ART in the horse. To answer this question, reproductive records of 289 mares bred b...
Sharp DC, McDowell KJ, Weithenauer J, Thatcher WW.Endometria from pregnant mares are able to produce PGF in vitro, but when co-incubated with conceptus membranes the amount and rate of PGF production is considerably reduced. To estimate the molecular weight of conceptus factors that inhibited PGF production, Day-14 conceptus membranes were placed inside bags constructed of dialysis tubing and co-incubated with endometria from Day-14 pregnant mares. PGF production was significantly reduced when membranes were in bags with molecular weight exclusion limits of 12,000, 6000, and 3500, but not of 1000, suggesting that conceptus PGF-inhibitory fact...
In this study, 198 donor mares of different breeds, ages, and reproductive category were inseminated with fresh, cooled and frozen or frozen and cooled semen at the embryo transfer station or in private artificial insemination centers during 10 breeding seasons. The results of this activity were retrospectively analyzed by Pearson Chi-square test and logistic regression to evaluate factors affecting multiple ovulations, embryo recovery, embryo quality, and embryo diameter. Out of the 661 cycles, 937 ovulations were recorded (mean ovulations/cycle: 1.42 ± 0.58). Ovulation rate and incidence of...
