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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.
Seasonal functional relevance of sperm characteristics in equine spermatozoa.
Theriogenology    January 18, 2010   Volume 73, Issue 7 950-958 doi: 10.1016/j.theriogenology.2009.11.023
Gamboa S, Rodrigues AS, Henriques L, Batista C, Ramalho-Santos J.A group of stallions with different reproductive indexes were used to study seasonal variations in sperm quality (Equus caballus). Semen samples were collected from late September to July and analyzed according to four seasonal periods: late September-December, January-March, late March-May, and June-July. Parameters monitored included sperm concentration, sperm motility, sperm morphology, sperm viability, acrosomal status, plasma membrane stability, and sperm mitochondrial membrane potential. Overall, seminal parameters monitored are affected mostly by time period, followed by animal and last...
Alkaline and acid phosphatase, β-glucuronidase and electrolyte levels in fractionated stallion ejaculates.
Reproduction in domestic animals = Zuchthygiene    January 16, 2010   Volume 45, Issue 6 e369-e374 doi: 10.1111/j.1439-0531.2009.01579.x
Kareskoski AM, Reilas T, Sankari S, Andersson M, Güvenc K, Katila T.Seminal plasma (SP) is a mixture of contents from the testes, epididymides and accessory sex glands. The sperm concentration is highest in the first few jets, or fractions, of the ejaculate, and the composition of SP varies between these fractions because accessory gland secretions are released in a specific order. The aim of this study was to compare the levels of Na, Cl, K, Mg, Ca, inorganic phosphate (Pi) and the enzymes alkaline phosphatase (AP), acid phosphatase (ACP) and β-glucuronidase (BG) in the different fractions of the ejaculate and in different stallions. All semen collections we...
Treatment of persistent mating-induced endometritis in mares with the non-steroid anti-inflammatory drug vedaprofen.
Reproduction in domestic animals = Zuchthygiene    January 16, 2010   Volume 45, Issue 6 e458-e460 doi: 10.1111/j.1439-0531.2009.01572.x
Rojer H, Aurich C.Recently, successful treatment of mares with a history of persistent mating-induced endometritis (PMIE) with dexamethasone has been reported. As systemic treatment of horses with glucocorticoids should be handled with caution, we tested the hypothesis that treatment with the non-steroid anti-inflammatory drug (NSAID) vedaprofen, an inhibitor of cyclooxygenase-2, may have comparative, positive effects on fertility. Barren mares with a history of repeated PMIE were treated with vedaprofen (n = 8; initially 2 mg/kg bodyweight followed by 1 mg/kg orally twice daily) from 1 day before the first ins...
Evaluation of shape variability of stallion sperm heads by means of image analysis and Fourier descriptors.
Animal reproduction science    December 16, 2009   Volume 119, Issue 1-2 50-55 doi: 10.1016/j.anireprosci.2009.12.007
Severa L, Máchal L, Svábová L, Mamica O.This study quantified and evaluated the variability of sperm head shape for 10 different stallions. Sperm head shape characteristics including sperm head length to width ratio, position of the center of gravity, curvature, and degree of roundness were assessed and analysed from images using elliptic Fourier descriptors and inverse Fourier transformation. The first four principal components accounted for 88.46-92.33% of the total variance and provided a good summary of the overall data. In the case of the ejaculate with defective sperm heads the components accounted for 97.35-98.21% of variatio...
Markers of the uterine innate immune response of the mare.
Animal reproduction science    December 3, 2009   Volume 119, Issue 1-2 31-39 doi: 10.1016/j.anireprosci.2009.11.008
Nash DM, Sheldon IM, Herath S, Lane EA.Reproductive efficiency in mares is low and persistent mating-induced endometritis (PMIE) is an important cause of subfertility. Mating-induced endometritis (MIE) an obligate precursor to PMIE, is a ubiquitous, transient inflammatory response to the presence of sperm, seminal components and pathogens. However, the specific inflammatory pathways that derive from MIE and that may also be precursors to PMIE are not clear. The ability to identify and measure robust, repeatable markers of inflammation integral to MIE may be key to understanding the progression to PMIE. The aim of the study was to (...
Effects of in vitro exposure to natural levels of zearalenone and its derivatives on chromatin structure stability in equine spermatozoa.
Theriogenology    December 1, 2009   Volume 73, Issue 3 392-403 doi: 10.1016/j.theriogenology.2009.09.023
Minervini F, Lacalandra GM, Filannino A, Nicassio M, Visconti A, Dell'Aquila ME.The purpose of this study was to assess the natural exposure of male horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate t...
Effects of inbreeding and other genetic components on equine fertility.
Animal : an international journal of animal bioscience    December 1, 2009   Volume 3, Issue 12 1662-1672 doi: 10.1017/S1751731109990553
Sairanen J, Nivola K, Katila T, Virtala AM, Ojala M.The Finnish mating records of Standardbred trotters (SB; n = 33 679) and Finnhorses (FH; n = 32 731) were analysed to study the effect of the level of inbreeding on foaling rates and to estimate the heritability of foaling rate. A linear mixed model was assumed, with the outcome of the foaling (foal or no foal) as the trait of the study. A restricted maximum likelihood-based method was used to calculate the estimates of the variance components. Predictions of breeding values and estimates of fixed effects were also calculated. The average level of inbreeding was 9.9% in the SB and 3.6% in the ...
Successful transfer of day 10 horse embryos: influence of donor-recipient asynchrony on embryo development.
Reproduction (Cambridge, England)    November 30, 2009   Volume 139, Issue 3 575-585 doi: 10.1530/REP-09-0306
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...
Post natal oestrogen administration stimulates precocious endometrial gland development in the horse.
Equine veterinary journal    November 26, 2009   Volume 41, Issue 7 678-684 doi: 10.2746/042516409x429428
Wilsher S, Lefranc AC, Allen WR.Fillies completely devoid of endometrial glands (uterine gland knockout; UGKO) would make ideal experimental models in which to study the role of endometrial histotroph in embryogenesis and early fetal development in the mare. Objective: Administration of a synthetic progestagen plus oestrogen to newborn filly foals and, thereafter, at regular intervals to age 6 months, would permanently suppress endometrial gland development. Methods: Nine half-sister Thoroughbred filly foals were treated, in 3 groups, with: A) the weakly active progestagen, norgestomet, administered from birth to age 6 month...
The secretions of oviduct epithelial cells increase the equine in vitro fertilization rate: are osteopontin, atrial natriuretic peptide A and oviductin involved?
Reproductive biology and endocrinology : RB&E    November 19, 2009   Volume 7 129 doi: 10.1186/1477-7827-7-129
Mugnier S, Kervella M, Douet C, Canepa S, Pascal G, Deleuze S, Duchamp G, Monget P, Goudet G.Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse. Results: In the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophor...
Immediate and delayed (after cooling) effects of centrifugation on equine sperm.
Theriogenology    November 17, 2009   Volume 73, Issue 2 225-231 doi: 10.1016/j.theriogenology.2009.09.003
Len JA, Jenkins JA, Eilts BE, Paccamonti DL, Lyle SK, Hosgood G.The objectives of this study were to determine the effects of centrifugation on equine sperm total and progressive motility, viability, and acrosomal integrity. We hypothesized that although high centrifugation forces would be detrimental to equine Equus caballus sperm, recovery rates would increase. Ejaculates from six stallions were collected, extended to a concentration of 25x10(6) cells/mL, and subjected for 10min to (1) no centrifugation (NC) or (2) centrifugation at 400xg, (3) 900xg, or (4) 4500xg. Before and after centrifugation (Day 0), and after 24h of cooling (Day 1), sperm motility ...
Evaluation of prolactin receptor (PRLR) as candidate gene for male fertility in Hanoverian warmblood horses.
Reproduction in domestic animals = Zuchthygiene    October 23, 2009   Volume 45, Issue 5 e124-e130 doi: 10.1111/j.1439-0531.2009.01533.x
Giesecke K, Hamann H, Sieme H, Distl O.Stallion fertility has increasing importance as the artificial insemination is employed in horses more intensely. Molecular genetic markers may be useful tools to evaluate the stallion fertility before breeding. The prolactin receptor gene (PRLR) was chosen as a candidate for stallion fertility because of its influence on testicular and accessory sex gland function. Screening the equine PRLR gene for polymorphisms in Hanoverian stallions revealed two single nucleotide polymorphisms (SNPs). Association and haplotype analyses were performed in 162 Hanoverian warmblood stallions for these intrage...
Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob) and leptin receptor (ObR) proteins in the horse.
Reproductive biology and endocrinology : RB&E    October 16, 2009   Volume 7 113 doi: 10.1186/1477-7827-7-113
Lange Consiglio A, Dell'Aquila ME, Fiandanese N, Ambruosi B, Cho YS, Bosi G, Arrighi S, Lacalandra GM, Cremonesi F.The identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity ...
Zonadhesin D3-polypeptides vary among species but are similar in Equus species capable of interbreeding.
Biology of reproduction    September 30, 2009   Volume 82, Issue 2 413-421 doi: 10.1095/biolreprod.109.077891
Tardif S, Brady HA, Breazeale KR, Bi M, Thompson LD, Bruemmer JE, Bailey LB, Hardy DM.Zonadhesin is a rapidly evolving protein in the sperm acrosome that confers species specificity to sperm-zona pellucida adhesion. Though structural variation in zonadhesin likely contributes to its species-specific function, the protein has not previously been characterized in organisms capable of interbreeding. Here we compared properties of zonadhesin in several animals, including the horse (Equus caballus), donkey (E. asinus), and Grevy's zebra (E. grevyi) to determine if variation in zonadhesin correlates with ability of gametes to cross-fertilize. Zonadhesin localized to the apical acroso...
The effect of hormone treatments (hCG and cloprostenol) and season on the incidence of hemorrhagic anovulatory follicles in the mare: a field study.
Theriogenology    September 23, 2009   Volume 72, Issue 9 1262-1267 doi: 10.1016/j.theriogenology.2009.07.022
Cuervo-Arango J, Newcombe JR.The association between use of hormone treatments to induce estrus and ovulation and the incidence of hemorrhagic anovulatory follicles (HAFs) was studied in a mixed population of mares (Equus caballus) during two breeding seasons in a commercial breeding clinic. Mares treated with cloprostenol (CLO) were more likely to develop HAFs than were mares with spontaneous cycles (P0.05) from that of mares with ovulatory cycles (10.5+/-1.5 yr).
Sexual behavior and ejaculate characteristics in Pêga donkeys (Equus asinus) mounting estrous horse mares (Equus caballus).
Theriogenology    September 22, 2009   Volume 73, Issue 1 56-63 doi: 10.1016/j.theriogenology.2009.07.026
Canisso IF, Carvalho GR, Morel MC, Guimarães JD, McDonnell SM.The objectives were to (i) characterize sexual behavior of donkey stallions (jacks; Equus asinus) during on-farm semen collection using estrous horse mares (mares; Equus caballus); (ii) compare behavior of young (less experienced) versus older (more experienced) jacks; (iii) determine whether semen suitable for artificial insemination (AI) could be collected using mares; and (iv) determine the suitability of using mares in field collection of semen from jacks. Six Pêga jacks (3.5 to 16 yr old), previously conditioned to breed mares, were used. Mount mares were confirmed in estrus by a teaser ...
Infertility and candidate gene markers for fertility in stallions: a review.
Veterinary journal (London, England : 1997)    August 26, 2009   Volume 185, Issue 3 265-271 doi: 10.1016/j.tvjl.2009.07.024
Giesecke K, Sieme H, Distl O.Stallion fertility is of high economic importance for the horse industry. The discovery of molecular mechanisms affecting fertility will be facilitated by the horse genome assembly and the development of novel tools for analysing complex genetic traits. Genetic markers in candidate genes, such as CRISP3, SPATA1 and INHBA, in breeding stallions have been associated with pregnancy rate per oestrus in mares. This paper reviews candidate autosomal, X and Y genes for stallion fertility, including genes encoding hormones and their receptors of the hypothalamic-pituitary axis, proteins of the seminal...
Cholesterol addition protects membrane intactness during cryopreservation of stallion sperm.
Animal reproduction science    August 25, 2009   Volume 118, Issue 2-4 194-200 doi: 10.1016/j.anireprosci.2009.08.011
Oliveira CH, Vasconcelos AB, Souza FA, Martins-Filho OA, Silva MX, Varago FC, Lagares MA.Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen-thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 x 10(6)spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 degrees C, and cryopreserved in...
The first ovulation of the breeding season in the mare: the effect of progesterone priming on pregnancy rate and breeding management (hCG response rate and number of services per cycle and mare).
Animal reproduction science    August 21, 2009   Volume 118, Issue 2-4 265-269 doi: 10.1016/j.anireprosci.2009.08.008
Cuervo-Arango J, Clark A.The mare is a seasonally polyestrus breeder. In early spring, the mare enters a "transition period" between the anovulatory season and the first ovulation of the year. This period is characterized by irregular estrus cycles and high incidence of regressing dominant follicles. There is a belief that pregnancy rates resulting from the first ovulation of the season is lower than in subsequent ovulations, however this has never been studied critically. Progestagens are often used as an aid to manage the transition period. The objective of this study was to compare pregnancy rates of mares from the...
Cholesterol-loaded-cyclodextrins and fertility potential of stallions spermatozoa.
Animal reproduction science    August 15, 2009   Volume 118, Issue 2-4 255-264 doi: 10.1016/j.anireprosci.2009.08.001
Spizziri BE, Fox MH, Bruemmer JE, Squires EL, Graham JK.Irreversible damage occurs to spermatozoal membranes, during the phase transition, when spermatozoa are cooled from room temperature to 5 degrees C. Some of this damage can be ameliorated by adding cholesterol to the membrane, thereby altering membrane lipid composition. Adding cholesterol-loaded cyclodextrins (CLCs) to stallion spermatozoa prior to freezing, increases cell cryosurvival. However, the fertilizing potential of CLC-treated stallion spermatozoa is unknown. To address this, experiments were conducted which evaluated the ability of CLC-treated stallion spermatozoa to capacitate, acr...
Clinical and subclinical endometritis in the mare: both threats to fertility.
Reproduction in domestic animals = Zuchthygiene    August 13, 2009   Volume 44 Suppl 3 10-22 doi: 10.1111/j.1439-0531.2009.01485.x
LeBlanc MM, Causey RC.Endometritis, a major cause of mare infertility arising from failure to remove bacteria, spermatozoa and inflammatory exudate post-breeding, is often undiagnosed. Defects in genital anatomy, myometrial contractions, lymphatic drainage, mucociliary clearance, cervical function, plus vascular degeneration and inflamm-ageing underlie susceptibility to endometritis. Diagnosis is made through detecting uterine fluid, vaginitis, vaginal discharge, short inter-oestrous intervals, inflammatory uterine cytology and positive uterine culture. However, these signs may be absent in subclinical cases. Hyper...
Does the microbial flora in the ejaculate affect the freezeability of stallion sperm?
Reproduction in domestic animals = Zuchthygiene    August 6, 2009   Volume 44, Issue 3 518-522 doi: 10.1111/j.1439-0531.2008.01267.x
Ortega-Ferrusola C, González-Fernández L, Muriel A, Macías-García B, Rodríguez-Martínez H, Tapia JA, Alonso JM, Peña FJ.In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze,three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were:Staphylococcus spp. and Micrococcus spp. (in all the stallions), beta-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3-5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in sta...
Cloprostenol in equine reproductive practice: something more than a luteolytic drug.
Reproduction in domestic animals = Zuchthygiene    August 5, 2009   Volume 45, Issue 5 e8-e11 doi: 10.1111/j.1439-0531.2009.01508.x
Cuervo-Arango J, Newcombe JR.Prostaglandin F(2α) and its analogues (PGF) are widely used in equine reproductive practice. The interval from PGF treatment to ovulation (ITO) varies greatly with a range from 2 to 16 days. Clinical observation suggests that mares mated and ovulated soon after PGF treatment may have poor fertility. Reproductive records of 329 cyclic Thoroughbred mares were analysed retrospectively. The following parameters were analysed: (i) use of cloprostenol; (ii) ITO and (iii) number of ovulations per cycle. According to these parameters, mares were classified into four groups. (i) mares with spontaneous...
Identification of chromosome abnormalities in the horse using a panel of chromosome-specific painting probes generated by microdissection.
Acta veterinaria Hungarica    July 29, 2009   Volume 57, Issue 3 369-381 doi: 10.1556/AVet.57.2009.3.3
Bugno M, Słota E, Pieńkowska-Schelling A, Schelling C.Fluorescent in situ hybridisation (FISH) using a panel of molecular probes for all chromosome pairs obtained by chromosome microdissection of the domestic horse ( Equus caballus ) was used to diagnose karyotype abnormalities in 35 horses (32 mares, 2 stallions and 1 intersex), which were selected for the study due to infertility (23 horses), reduced fertility (10 horses) and developmental anomalies (2 horses). The use of the FISH technique with probes for each horse chromosome pair enabled the diagnosis of many different chromosome aberrations in this population. Among the horses analysed, 21 ...
Effect of cryopreservation on nitric oxide production by stallion spermatozoa.
Biology of reproduction    July 15, 2009   Volume 81, Issue 6 1106-1111 doi: 10.1095/biolreprod.109.078220
Ortega Ferrusola C, González Fernández L, Macías García B, Salazar-Sandoval C, Morillo Rodríguez A, Rodríguez Martinez H, Tapia JA, Peña FJ.The ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe, 4,5-diaminofluorescenin diacetate. The presence of NO synthase (NOS) was investigated by Western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS antibodies (Abs). While NO was detected both in fresh and FT sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 Ab intensively labeled a single band with an app...
Effect of day of transfer and treatment administration on the recipient on pregnancy rates after equine embryo transfer.
Veterinary research communications    July 14, 2009   Volume 33 Suppl 1 113-116 doi: 10.1007/s11259-009-9303-7
Panzani D, Crisci A, Rota A, Camillo F.No abstract available
Single-layer centrifugation with Androcoll-E can be scaled up to allow large volumes of stallion ejaculate to be processed easily.
Theriogenology    July 14, 2009   Volume 72, Issue 6 879-884 doi: 10.1016/j.theriogenology.2009.05.015
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...
Influence of seminal plasma on fertility of fresh and frozen-thawed stallion epididymal spermatozoa.
Animal reproduction science    July 14, 2009   Volume 118, Issue 1 48-53 doi: 10.1016/j.anireprosci.2009.06.009
Heise A, Kähn W, Volkmann DH, Thompson PN, Gerber D.The use of epididymal stallion spermatozoa for routine artificial insemination can secure easy future use of valuable genetics after unforeseen death or injury of a valuable stallion. The aims of this study were to (1) directly compare pregnancy rates for fresh and frozen-thawed stallion epididymal and ejaculated spermatozoa after conventional artificial insemination and (2) to investigate the effect of seminal plasma on the fertility of epididymal spermatozoa after insemination. Twenty-one mares were randomly assigned to three stallions. Mares were inseminated at five consecutive oestrous per...
New insights into the mechanisms of fertilization: comparison of the fertilization steps, composition, and structure of the zona pellucida between horses and pigs.
Biology of reproduction    July 8, 2009   Volume 81, Issue 5 856-870 doi: 10.1095/biolreprod.109.077651
Mugnier S, Dell'Aquila ME, Pelaez J, Douet C, Ambruosi B, De Santis T, Lacalandra GM, Lebos C, Sizaret PY, Delaleu B, Monget P, Mermillod P....The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was ...
Freezing equine semen: the effect of combinations of semen extenders and glycerol on post-thaw motility.
Australian veterinary journal    July 4, 2009   Volume 87, Issue 7 275-279 doi: 10.1111/j.1751-0813.2009.00452.x
Scherzer J, Fayrer-Hosken RA, Aceves M, Hurley DJ, Ray LE, Jones L, Heusner GL.We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility. Methods: A randomised 2 x 3 block design was used. Methods: Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy...
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