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Topic:Reproduction

Reproduction in horses encompasses the biological processes and mechanisms involved in the breeding and development of equine offspring. This includes the study of reproductive anatomy, physiology, and endocrinology in both mares and stallions. Key areas of interest include the estrous cycle, ovulation, conception, gestation, and parturition. Researchers also examine factors influencing fertility, reproductive technologies such as artificial insemination and embryo transfer, and management practices that impact reproductive success. This page compiles peer-reviewed research studies and scholarly articles that explore the physiological, genetic, and environmental aspects of equine reproduction.
Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) follicular signalling is conserved in the mare ovary.
Reproduction, fertility, and development    September 26, 2017   Volume 30, Issue 4 624-633 doi: 10.1071/RD17024
Hall SE, Upton RMO, McLaughlin EA, Sutherland JM.The mare ovary is unique in its anatomical structure; however, the signalling pathways responsible for physiological processes, such as follicular activation, remain uncharacterised. This provided us with the impetus to explore whether signalling molecules from important folliculogenesis pathways, phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT), are conserved in the mare ovary. Messenger RNA expression of six genes important in follicle development was measured using quantitative polymerase chain reaction and pr...
Morphological evaluation of Day 8 embryos developed during induced aluteal cycles in the mare.
Theriogenology    September 25, 2017   Volume 105 178-183 doi: 10.1016/j.theriogenology.2017.09.029
Leisinger CA, Medina V, Markle ML, Paccamonti DL, Pinto CRF.A novel in vivo model utilizing serial administrations of PGF2α to induce aluteal cycles in the mare was used to evaluate the effects of progesterone-deprivation on the morphology of in vivo preimplantation embryos. We hypothesized that equine embryos produced during induced aluteal cycles (AL) would be developmentally affected, characterized by earlier embryo stage at collection, smaller embryo diameter, and lower quality grade, compared with those collected on the same day post-ovulation from control cycles during diestrus (high progesterone; > 4 ng/mL). Seven cyclic mares with a median ...
A Brief Account of the Discovery of the Fetal/Placental Unit for Estrogen Production in Equine and Human Pregnancies: Relation to Human Medicine.
The Yale journal of biology and medicine    September 25, 2017   Volume 90, Issue 3 449-461 
Raeside JI.The role of steroids in human medicine is well recognized, but the major contributions made by the large domestic animals as a source of material in the discovery, isolation, and determination of the structure of the steroid hormones is less well appreciated. After a brief reminder of the early efforts to obtain a reliable source of steroids for clinical use, the narrative here is to outline one example where success was ultimately achieved for estrogen replacement therapy. Whereas knowledge of the high concentrations of estrogens in urine of pregnant women and mares dates from the late 1920s,...
An improved vitrification protocol for equine immature oocytes, resulting in a first live foal.
Equine veterinary journal    September 21, 2017   Volume 50, Issue 3 391-397 doi: 10.1111/evj.12747
Ortiz-Escribano N, Bogado Pascottini O, Woelders H, Vandenberghe L, De Schauwer C, Govaere J, Van den Abbeel E, Vullers T, Ververs C, Roels K....The success rate for vitrification of immature equine oocytes is low. Although vitrified-warmed oocytes are able to mature, further embryonic development appears to be compromised. Objective: The aim of this study was to compare two vitrification protocols, and to examine the effect of the number of layers of cumulus cells surrounding the oocyte during vitrification of immature equine oocytes. Methods: Experimental in vitro and in vivo trials. Methods: Immature equine oocytes were vitrified after a short exposure to high concentrations of cryoprotective agents (CPAs), or a long exposure to l...
Comparison of physical body growth and metabolic and reproductive endocrine functions between north and south climates of Japan in trained Thoroughbred yearling horses.
Journal of equine science    September 20, 2017   Volume 28, Issue 3 77-86 doi: 10.1294/jes.28.77
Tangyuenyong S, Sato F, Nambo Y, Murase H, Endo Y, Tanaka T, Nagaoka K, Watanabe G.This study aimed to compare body growth, metabolic, and reproductive hormonal changes in trained Thoroughbred yearling horses under different climate conditions with and without light supplementation (LS). Thoroughbred yearlings raised at research centers of the Japan Racing Association in Hokkaido (north) or Miyazaki (south) were divided into control and LS groups. In the LS groups, 44 colts and 47 fillies from Hokkaido and 11 colts and 11 fillies from Miyazaki were exposed to LS with an extended photoperiod of 14.5 hr of daylight and 9.5 hr of darkness. One week before and once a month after...
Exposure to follicular fluid during oocyte maturation and oviductal fluid during post-maturation does not improve in vitro embryo production in the horse.
Zygote (Cambridge, England)    September 20, 2017   Volume 25, Issue 5 612-630 doi: 10.1017/S096719941700048X
Douet C, Parodi O, Martino NA, Lacalandra GM, Nicassio M, Reigner F, Deleuze S, Dell'Aquila ME, Goudet G.Most wild equids and many domestic horse breeds are at risk of extinction, so there is an urgent need for genome resource banking. Embryos cryopreservation allows the preservation of genetics from male and female and is the fastest method to restore a breed. In the equine, embryo production in vitro would allow the production of several embryos per cycle. Intracytoplasmic sperm injection (ICSI) is used to generate horse embryos, but it requires expensive equipment and expertise in micromanipulation, and blastocyst development rates remain low. No conventional in vitro fertilization (IVF) techn...
Lipidomics of equine amniotic fluid: Identification of amphiphilic (O-acyl)-ω-hydroxy-fatty acids.
Theriogenology    September 20, 2017   Volume 105 120-125 doi: 10.1016/j.theriogenology.2017.09.012
Wood PL, Ball BA, Scoggin K, Troedsson MH, Squires EL.Amniotic fluid is essential for the growth and maturation of the fetus prior to parturition. While our knowledge of human amniotic fluid is extensive, current data for equine amniotic fluid is limited. We therefore undertook a detailed lipidomics analysis of equine amniotic fluid. Using a non-targeted high-resolution mass spectrometric lipidomics analysis of equine amniotic fluid, we were able to characterize a diverse array of individual lipids. This non-biased analytical approach detected, for the first time, the presence of (O-acyl)-ω-hydroxy-fatty acids (OAHFA) with up to 52 carbon chain ...
Measurement of urinary concentrations of the mycotoxins zearalenone and sterigmatocystin as biomarkers of exposure in mares.
Reproduction in domestic animals = Zuchthygiene    September 18, 2017   Volume 53, Issue 1 68-73 doi: 10.1111/rda.13054
Takagi M, Uno S, Kokushi E, Sato F, Wijayagunawardane M, Fink-Gremmels J.Mycotoxins may affect animal health, including reproduction. Little is known about the clinical relevance of exposure of horses to contaminated feed. This study aimed at (i) monitoring the levels of the mycotoxins zearalenone (ZEN), with its metabolites α- and β-zearalenol (α- and β-ZOL), and sterigmatocystin (STC) in urine samples from thoroughbred mares in Japan and (ii) relating these findings to the potential effects on reproductive efficacy of breeding mares. Sixty-three urine samples of breeding mares from 59 breeding farms were used. Urine samples and reproductive records were colle...
Pre-ovulatory follicle affects corpus luteum diameter, blood flow, and progesterone production in mares.
Animal reproduction science    September 8, 2017   Volume 187 1-12 doi: 10.1016/j.anireprosci.2017.09.003
Ishak GM, Bashir ST, Gastal MO, Gastal EL.Color Doppler ultrasonography was used to study the temporal relationships between pre-ovulatory follicle (POF) and corpus luteum (CL) diameter and blood flow, with systemic progesterone (P4) concentration during two transitional ovulatory seasons in mares. Variables of POF and CL/P4 were evaluated for 6days before and 17days after ovulation, respectively. Evaluations were performed during two consecutive estrous cycles in spring and fall seasons, and during the last estrous cycle of the season. There were significant correlations among POF and CL variables, and P4 concentration that ranged fr...
Equine fetal adrenal, gonadal and placental steroidogenesis.
Reproduction (Cambridge, England)    September 8, 2017   Volume 154, Issue 4 445-454 doi: 10.1530/REP-17-0239
Legacki EL, Ball BA, Corbin CJ, Loux SC, Scoggin KE, Stanley SD, Conley AJ.Equine fetuses have substantial circulating pregnenolone concentrations and thus have been postulated to provide significant substrate for placental 5α-reduced pregnane production, but the fetal site of pregnenolone synthesis remains unclear. The current studies investigated steroid concentrations in blood, adrenal glands, gonads and placenta from fetuses (4, 6, 9 and 10 months of gestational age (GA)), as well as tissue steroidogenic enzyme transcript levels. Pregnenolone and dehydroepiandrosterone (DHEA) were the most abundant steroids in fetal blood, pregnenolone was consistently higher bu...
Reproductive careers of Thoroughbred broodmares before and after surgical correction of ≥360 degree large colon volvulus.
Equine veterinary journal    September 7, 2017   Volume 50, Issue 2 208-212 doi: 10.1111/evj.12736
Leahy ER, Holcombe SJ, Hackett ES, Scoggin CF, Embertson RM.Limited data exist describing broodmare longevity and reproductive efficiency after surgical correction of ≥360 degree large colon volvulus (° LCV). Objective: Compare career duration and foals delivered for broodmares before and after ≥360° LCV surgery. Methods: Retrospective case series. Methods: Broodmares registered with The Jockey Club that had surgical correction of ≥360° LCV and survived to hospital discharge at Rood and Riddle Equine Hospital from 1 January 2000 to 31 December 2015 were included. Information was collected from the hospital's medical data base and The Jockey Cl...
A lectin histochemical study to detect variation in glycosylation at the feto-maternal interface in three interbreeding equine species.
Placenta    September 4, 2017   Volume 58 115-121 doi: 10.1016/j.placenta.2017.08.075
Jones CJP, Allen WRT, Wilsher S.In this study, we compare glycosylation at the fetomaternal interface in 3 equine species: horse, donkey and zebra, all of which can interbreed to produce hybrids, to assess their glycan similarities and differences. Methods: Sections cut from 3 specimens of horse (Equus caballus) placenta (50, 200 and 280 days gestation), one donkey (Equus asinus) placenta (65 cm crown-rump length) and 5 specimens of zebra (Equus quagga) placentae (81-239 days gestation) were stained with a panel of 24 biotinylated lectins using an avidin-peroxidase revealing system. Results: There were only slight quantitat...
Serum amyloid A protein (SAA), haptoglobin (Hp) and selected hematological and biochemical parameters in wild mares before and after parturition.
Polish journal of veterinary sciences    September 3, 2017   Volume 20, Issue 2 299-305 doi: 10.1515/pjvs-2017-0036
Krakowski L, Bartoszek P, Krakowska I, Olcha P, Piech T, Stachurska A, Brodzki P.The aim of the study was to evaluate physiological changes in hematological and biochemical parameters in mares in perinatal period. Blood samples were collected from 24 pregnant Polish Konik breed mares which were divided into two groups. The first group (Group - I, n=12) comprised mares living in the wild, in the reserve. The second group (Group - II, n=12) consisted of mares kept in stables. The blood was collected 2 weeks prior to the parturition, then 24 hours after the delivery, and then at the 7th and 21st day after foaling. When comparing the two groups before the parturition, no signi...
Theriogenology Question of the Month.
Journal of the American Veterinary Medical Association    September 1, 2017   Volume 251, Issue 6 657-660 doi: 10.2460/javma.251.6.657
Boye JK, Byrne BA, Alex CE, Affolter VK, Christensen BW, Dujovne GA.No abstract available
Likelihood of pregnancy after embryo transfer is reduced in recipient mares with a short preceding oestrus.
Equine veterinary journal    September 1, 2017   Volume 50, Issue 3 386-390 doi: 10.1111/evj.12739
Cuervo-Arango J, Claes AN, Ruijter-Villani M, Stout TA.Previous surveys reported a positive association between the length of the follicular phase and subsequent fertility in embryo transfer donor and Thoroughbred mares. However, it is unclear whether a longer oestrus positively influences fertilisation and oviductal development (oocyte quality, oviductal environment), or uterine receptivity and survival of the embryo in the uterus. Objective: To determine the effect of length of oestrus (characterised by duration of endometrial oedema) on likelihood of pregnancy and early embryo loss (EEL) in recipient mares after embryo transfer (ET). Methods: R...
Association of the cysteine-rich secretory protein-3 (CRISP-3) and some of its polymorphisms with the quality of cryopreserved stallion semen.
Reproduction, fertility, and development    September 1, 2017   Volume 30, Issue 3 563-569 doi: 10.1071/RD17044
Usuga A, Rojano BA, Restrepo G.Contribution of seminal plasma proteins to semen freezability has been reported in several species, suggesting these proteins as genetic markers. The aim of this study was to evaluate the relationship between cysteine-rich secretory protein-3 (CRISP-3) and some of its single-nucleotide polymorphisms (SNPs) with post-thawing semen quality in stallions. DNA was obtained from 100 stallions, regions of interest were amplified by polymerase chain reaction and sequenced. Evaluated SNPs within the equine CRISP-3 gene were CRISP3c.+199A>G (SNP1), CRISP3c.+566C>A (SNP2), CRISP3c.+622G>A (SNP3)...
D-loop haplotype diversity in Brazilian horse breeds.
Genetics and molecular biology    August 31, 2017   Volume 40, Issue 3 604-609 doi: 10.1590/1678-4685-GMB-2016-0166
Ianella P, Albuquerque MSM, Paiva SR, Egito AA, Almeida LD, Sereno FTPS, Carvalho LFR, Mariante AS, McManus CM.The first horses were brought to Brazil by the colonizers after 1534. Over the centuries, these animals evolved and adapted to local environmental conditions usually unsuitable for exotic breeds, thereby originating locally adapted Brazilian breeds. The present work represents the first description of maternal genetic diversity in these horse breeds based on D-loop sequences. A D-Loop HSV-I fragment of 252 bp, from 141 horses belonging to ten Brazilian breeds / genetic groups (locally adapted and specialized breeds) were analysed. Thirty-five different haplotypes belonging to 18 haplogroups we...
Milk-fat globule epidermal growth factor 8 (MFGE8) is expressed at the embryo- and fetal-maternal interface in equine pregnancy.
Reproduction, fertility, and development    August 30, 2017   Volume 30, Issue 4 585-590 doi: 10.1071/RD17094
Barua S, Macedo A, Kolb DS, Wynne-Edwards KE, Klein C.Milk-fat globule epidermal growth factor (EGF) 8 protein (MFGE8), also known as lactadherin, promotes cell adhesion in an Arg-Gly-Asp (RGD)-dependent modus via integrins. In the present study, the expression of MFGE8 was examined in equine endometrium during oestrus and at Days 12 and 16 after ovulation in pregnant and non-pregnant mares and in mares during the 5th month of gestation. Results demonstrated that MFGE8 is expressed at the embryo- and fetal-maternal interface in equine pregnancy. In non-pregnant endometrium its expression was upregulated by oestrogen, a finding that was confirmed ...
RNA Interference and BMP-2 Stimulation Allows Equine Chondrocytes Redifferentiation in 3D-Hypoxia Cell Culture Model: Application for Matrix-Induced Autologous Chondrocyte Implantation.
International journal of molecular sciences    August 24, 2017   Volume 18, Issue 9 1842 doi: 10.3390/ijms18091842
Rakic R, Bourdon B, Hervieu M, Branly T, Legendre F, Saulnier N, Audigié F, Maddens S, Demoor M, Galera P.As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microe...
Innate immune genes in persistent mating-induced endometritis in horses.
Reproduction, fertility, and development    August 24, 2017   Volume 30, Issue 3 533-545 doi: 10.1071/RD17157
Marth CD, Firestone SM, Hanlon D, Glenton LY, Browning GF, Young ND, Krekeler N.Persistent mating-induced endometritis (PMIE) severely decreases fertility in horses. The aim of the present study was to evaluate differences between horses susceptible to PMIE and a control group in terms of the expression of selected immune response and effector genes, and the effects of oestrous cycle stage on this expression. Endometrial biopsies from 18 uterine samples of mares in the control group (eight in dioestrus, 10 in oestrus) and 16 PMIE-susceptible mares (four in dioestrus, 12 in oestrus) were analysed by quantitative real-time reverse transcription-polymerase chain reaction. Ge...
Horse spermatogonial stem cell cryopreservation: feasible protocols and potential biotechnological applications.
Cell and tissue research    August 22, 2017   Volume 370, Issue 3 489-500 doi: 10.1007/s00441-017-2673-1
Costa GMJ, Avelar GF, Lacerda SMSN, Figueiredo AFA, Tavares AO, Rezende-Neto JV, Martins FGP, França LR.The establishment of proper conditions for spermatogonial stem cells (SSCs) cryopreservation and storage represents an important biotechnological approach for the preservation of the genetic stock of valuable animals. This study demonstrates the effects of different cryopreservation protocols on the survival rates and phenotypic expression of SSCs in horses. The cells were enzymatically isolated from testes of eight adult horses. After enrichment and characterization of germ cells in the suspension, the feasibility of several cryopreservation protocols were evaluated. Three different cryomedia...
Testicular volumetry and prediction of daily sperm output in stallions by orchidometry and two- and three-dimensional sonography.
Theriogenology    August 19, 2017   Volume 104 149-155 doi: 10.1016/j.theriogenology.2017.08.015
Pricking S, Bollwein H, Spilker K, Martinsson G, Schweizer A, Thomas S, Oldenhof H, Sieme H.Accurate determination of the testes volume and prediction of the daily sperm output (DSO) is valuable information for reproductive management of a stallion. The aim of this study was to compare different methods for measuring the testes volume, including caliper, 2D and 3D ultrasound. Special emphasis was on feasibility of 3D volume analysis. First, 22 castrated testes were measured and derived volumes were compared with volumes determined via volume displacement in a graded cylinder with saline solution. Then, during the breeding season, testes sizes of 52 stallions were measured in vivo an...
Inflammation and fertility in the mare.
Reproduction in domestic animals = Zuchthygiene    August 18, 2017   Volume 52 Suppl 3 14-20 doi: 10.1111/rda.13013
Christoffersen M, Troedsson M.A transient uterine inflammation post-breeding is a normal physiological reaction in the mare, and it is believed that the inflammatory response is necessary to eliminate bacteria and excess spermatozoa introduced into the uterine lumen. A tight balance between multiple pro- and anti-inflammatory factors is required for resolving the breeding-induced inflammation within 24-36 hr in the reproductively healthy mare, whereas a subpopulation of mares is susceptible to development of a persistent infection that can interfere with fertility. The aetiology of persistent endometritis can be either ba...
Redox cycling induces spermptosis and necrosis in stallion spermatozoa while the hydroxyl radical (OH•) only induces spermptosis.
Reproduction in domestic animals = Zuchthygiene    August 17, 2017   Volume 53, Issue 1 54-67 doi: 10.1111/rda.13052
Martín Muñoz P, Anel-López L, Ortiz-Rodríguez JM, Álvarez M, de Paz P, Balao da Silva C, Rodríguez Martinez H, Gil MC, Anel L, Peña FJ....Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2-methyl-1,4-naphthoquinone (menadione), or hydroxyl radical formation after FeSO exposure. Either exposure induced significant increases (p < 0.05) in two marke...
Progress in broodmare practice as based on recent papers in Equine Veterinary Journal.
Equine veterinary journal    August 15, 2017   Volume 49, Issue 5 565-566 doi: 10.1111/evj.12714
Pycock JF.No abstract available
Evaluation of an intravaginal triptorelin acetate gel for inducing ovulation in mares.
Journal of animal science    August 15, 2017   Volume 95, Issue 8 3631-3638 doi: 10.2527/jas.2017.1373
Sinclair CD, Webel SK, Douthit TL, Murray LW, Jager AL, Grieger DM, Kouba JM.The objective of these studies was to investigate the efficacy of an intravaginal triptorelin acetate (TA) gel as an ovulation-inducing agent in mares. In Exp. 1, 24 mares were blocked using a combination of parity and age and randomly assigned to 1 of 3 treatment groups: 5 mL TA gel (TA5), 10 mL TA gel (TA10), or 5 mL vehicle gel only (CON). Following the appearance of a follicle ≥ 25 mm, a blood sample was obtained for measurement of LH from each mare every 24 h until treatment administration. When a follicle ≥ 35 mm was observed, treatments were administered intravaginally. Following tr...
Effect of housing system on reproductive behaviour and on some endocrinological and seminal parameters of donkey stallions.
Reproduction in domestic animals = Zuchthygiene    August 14, 2017   Volume 53, Issue 1 40-47 doi: 10.1111/rda.13050
Rota A, Sgorbini M, Panzani D, Bonelli F, Baragli P, Ille N, Gatta D, Sighieri C, Casini L, Maggiorelli MM, Aurich C, Camillo F.Reproductive management of male donkeys employed for artificial breeding has been poorly studied. The aim of this study was to evaluate the effect of housing system, with the animals grouped together in a paddock or kept in individual boxes, on sexual behaviour, cortisol and testosterone concentration and seminal characteristics of adult male donkeys. The study included four Amiata donkey jacks (stallions) from which ejaculates, saliva and blood were collected during two distinct 3 weeks periods, one in the group and one in the box housing system. Time needed for semen collection was shorter ...
Uterine clinical findings, fertility rate, leucocyte migration, and COX-2 protein levels in the endometrial tissue of susceptible mares treated with platelet-rich plasma before and after AI.
Theriogenology    August 12, 2017   Volume 104 120-126 doi: 10.1016/j.theriogenology.2017.08.007
Segabinazzi LG, Friso AM, Correal SB, Crespilho AM, Dell'Aqua JA, Miró J, Papa FO, Alvarenga MA.Persistent mating-induced endometritis (PMIE) results in decreased fertility in horses, thereby causing a significant impact in the horse market. Platelet-rich plasma (PRP), a modulator of the inflammatory response, has been largely used in veterinary medicine. Here, we investigated the effects of PRP on uterine inflammation, conception rate, endometrial polymorphonuclear neutrophil (PMN) migration, and COX-2 protein levels in the endometrial tissue. Thirteen PMIE-susceptible mares were used for artificial insemination (AI). The mares were inseminated with fresh semen in three consecutive cycl...
From Peptide Masses to Pregnancy Maintenance: A Comprehensive Proteomic Analysis of The Early Equine Embryo Secretome, Blastocoel Fluid, and Capsule.
Proteomics    August 8, 2017   Volume 17, Issue 17-18 doi: 10.1002/pmic.201600433
Swegen A, Grupen CG, Gibb Z, Baker MA, de Ruijter-Villani M, Smith ND, Stout TAE, Aitken RJ.Early pregnancy in the mare is a poorly understood, high risk period during which the embryo communicates its presence to the maternal endometrium. Remarkably, the maternal recognition of pregnancy signal is unknown in the horse. This study aimed to profile the proteins secreted by equine blastocysts into their immediate environment, along with proteins contained in the blastocoel and within the acellular embryo capsule. Embryos were recovered on day 8 after ovulation and cultured for 48 hours. Secretomes of day 9 and day 10 embryos were analyzed by LC-MS/MS and supported by analysis of blast...
Do different characteristics of two emasculators make a difference in equine castration?
Equine veterinary journal    August 1, 2017   Volume 50, Issue 1 141-144 doi: 10.1111/evj.12713
Comino F, Giusto G, Caramello V, Gandini M.The Serra and Reimer emasculators are frequently used in equine orchiectomy. They differ in jaw profile and the mechanism by which they achieve haemostasis. Objective: To investigate whether the haemostatic capacities of the Reimer and Serra emasculators in open and closed castration differ, to compare the haemostatic capacities of each emasculator in both open and closed castration, and to assess whether the tensile strength of the parietal tunic in closed castration differs according to whether a Reimer or Serra emasculator is used. Methods: Ex vivo randomised study. Methods: Eighty equine c...
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