Equid semen refers to the reproductive fluid produced by male horses, which contains sperm cells necessary for fertilization. The quality and viability of equid semen are important for successful breeding programs and can be influenced by various factors, including genetics, age, diet, and environmental conditions. Key parameters used to assess semen quality include volume, concentration, motility, and morphology of sperm cells. Techniques for collecting, evaluating, and preserving equid semen, such as cryopreservation, are critical for artificial insemination practices. This page compiles peer-reviewed research studies and scholarly articles that explore the physiology, assessment, and management of equid semen in the context of equine reproduction.
Lagares MA, Martins HS, Carvalho IA, Oliveira CA, Souza MR, Penna CF, Cruz BC, Stahlberg R, Henry MR.Extenders with a defined composition containing only components with clearly protective effects on sperm during storage would be an advantage. The aims of the present work were to assess whether caseinate, improves cooled and frozen equine semen quality. Semen from six stallions were suspended with four different cooling extenders C1) Kenney extender; C2) 0.6 % caseinate; C3) 2.7 % caseinate ; and C4) C1 + 2.1 % caseinate, and frozen extenders: F1) INRA 82 extender; F2) 1.35 % caseinate; and F3) 2.7 % caseinate. Although there was no significant difference between the motility rate among the c...
Lindahl J, Dalin AM, Stuhtmann G, Morrell JM.One of the challenges faced by equine breeders is ensuring delivery of good quality semen doses for artificial insemination when the mare is due to ovulate. Single Layer Centrifugation (SLC) has been shown to select morphologically normal spermatozoa with intact chromatin and good progressive motility from the rest of the ejaculate, and to prolong the life of these selected spermatozoa in vitro. The objective of the present study was a proof of concept, to determine whether fertilizing ability was retained in SLC-selected spermatozoa during prolonged storage. Results: Sixteen mares were insemi...
Mawyer JD, Cavinder CA, Vogelsang MM, Sigler DH, Love CC, Brinsko SP, Blanchard TL, Varner DD, Arnold CE, Teague S, Gordon RK.Stallions (n = 8) were implanted with a thermal sensory device in the muscle of the neck and the subcutaneous tissue of the scrotum and then assigned to either a nonexercise (Non-EX; n = 4) or exercise (EX; n = 4) group. A motorized equine exerciser was used to work EX stallions 30 min/d for 4 d/wk during a 12-wk period from July through October 2010. Temperatures (subcutaneous scrotal, intramuscular neck, and rectal) were recorded at 0, 22, and 30 min after the start of exercise, as well as 60 and 120 min post-exercise. Hourly ambient temperature and relative humidity data were also obtained....
Crespo F, Gosalvez J, Gutiérrez-Cepeda L, Serres C, Johnston SD.Stallion spermatozoa recovered and examined immediately after colloidal centrifugation resulted in a higher straight-line velocity (VSL) than sperm processed using direct conventional centrifugation (p = 0.000), but there was no differences in the progressive motility or sperm DNA fragmentation (SDF) as determined by the sperm chromatin dispersion assay. However, when centrifuged spermatozoa were incubated at 37 °C for 24 h to determine the rate of SDF (r-SDF), a lower r-SDF (p = 0.0011) was observed in those sperm recovered after colloidal separation (0.5 ± 0.1%/h) compared to direct (1.2 ...
Scarlet D, Budik S, Aurich C.A new device for storage and shipping of cell cultures--the Petaka G3 cell management device--was tested for its applicability for cooled-storage of equine semen. Semen from three stallions was processed with EquiPro extender either without antibiotics (three ejaculates per stallion) or with gentamicin (250 mg/l; three ejaculates per stallion). Semen was either stored at five (anaerobic conditions) or 15 °C (aerobic conditions) in syringes or cell culture devices. Total and progressive motility, as well as membrane integrity of spermatozoa, were evaluated from days 1 to 7 after collection wit...
Vidament M, Magistrini M, Le Foll Y, Levillain N, Yvon JM, Duchamp G, Blesbois E.This study tested whether variable temperatures (from -0.5 to 15 °C) and air exposure could be used under laboratory and under field conditions to store stallion sperm diluted in extender INRA96 without loss of fertility. Experiment 1 (laboratory conditions) measured the effects of two 72 h storage conditions (5 °C with air vs. 15 °C without air). Experiment 2 (fixed field conditions) measured the effects of 22 h of storage without air in disposable containers maintained at four ambient temperatures (7 °C, 17 °C, 27 °C, 39 °C with semen at -0.5 °C to 3 °C, 4 °C to 7 °C, 8 °C to 10 ...
Barrier-Battut I, Bonnet C, Giraudo A, Dubois C, Caillaud M, Vidament M.Fertility is reduced after semen cooling for a considerable number of stallions. The main hypotheses include alterations in plasma membrane following cooling and deleterious influence of seminal plasma. However, interindividual variability is controversial. We hypothesized that the removal of seminal plasma could enhance motility in some 'poor cooler' stallions, but could also affect, negatively or positively, membrane quality in some stallions. This study examined the effect of centrifugation, followed or not by removal of seminal plasma, on parameters indicating semen quality after 48 h at 4...
Fayrer-Hosken R, Stanley A, Hill N, Heusner G, Christian M, De La Fuente R, Baumann C, Jones L.The cellular effects of tall fescue grass-associated toxic ergot alkaloids on stallion sperm and colt testicular tissue were evaluated. This was a continuation of an initial experiment where the effects of toxic ergot alkaloids on the stallion spermiogram were investigated. The only spermiogram parameter in exposed stallions that was affected by the toxic ergot alkaloids was a decreased gel-free volume of the ejaculate. This study examined the effect of toxic ergot alkaloids on chilling and freezing of the stallion sperm cells. The effect of toxic ergot alkaloids on chilled extended sperm cell...
Blanchard TL, Varner DD, Brinsko SP, Love CC.Determining the cause of failure to ejaculate sperm can be a diagnostic dilemma. The first diagnostic step is to ascertain whether the stallion is ejaculating. If the stallion appears to ejaculate, but there is azoospermia (absence of sperm in the seminal fluid), testing alkaline phosphatase (ALP) activity in seminal plasma can determine whether testicular and epididymal fluids are present. If ALP activity is low, the possibility of either blockage to sperm outflow in the excurrent duct system or retrograde ejaculation should be pursued diagnostically. If ALP activity is high, the possibility ...
Morrell JM, Pihl J, Dalin AM, Johannisson A.There is controversy about whether the presence of some seminal plasma (SP) in an equine insemination dose is necessary for promoting fertility. A new technique for improving stallion sperm quality, single layer centrifugation (SLC) using a species-specific colloid, Androcoll-E, selects a sperm subpopulation that is highly motile with normal morphology, intact membranes and good chromatin integrity from the rest of the ejaculate and removes SP. The present study was designed to investigate the effect of restoring homologous SP (5% and 10%) on the progressive motility, velocity, and chromatin i...
Oldenhof H, Friedel K, Akhoondi M, Gojowsky M, Wolkers WF, Sieme H.Stallion sperm exhibits great male-to-male variability in survival after cryopreservation. In this study, we have investigated if differences in sperm freezability can be attributed to membrane phase and permeability properties. Fourier transform infrared spectroscopy (FTIR) was used to determine supra and subzero membrane phase transitions and characteristic subzero membrane hydraulic permeability parameters. Sperm was obtained from stallions that show differences in sperm viability after cryopreservation. Stallion sperm undergoes a broad and gradual phase transition at suprazero temperatures...
de Andrade AF, Zaffalon FG, Celeghini EC, Nascimento J, Bressan FF, Martins SM, de Arruda RP.The objective was to verify the relationship between equine semen cryopreservation and changes related to increased lipid peroxidation. Also, addition of autologous or homologous seminal plasma from a stallion with a good freezing response to post-thawed sperm was tested to determine whether it would confer protection. Frozen-thawed sperm were evaluated and allocated into three groups: without plasma addition, and supplemented with either homologous or autologous seminal plasma. All groups were evaluated at 0, 60 and 120 min after incubation at 37 °C. Cryopreservation did not increase plasma ...
Coutinho da Silva MA, Seidel GE, Squires EL, Graham JK, Carnevale EM.The effects of semen extender components on the ability of stallion sperm to bind to the zona pellucida (ZP) and the suitability of using bovine ZP for a ZP-binding assay for stallion sperm were investigated in a series of experiments. In Experiment I, binding of stallion sperm to both bovine and equine ZP was significantly increased when a skim milk-based extender (EZM) was used. In Experiment II, a threefold increase in sperm binding to ZP was observed when sperm were diluted in EZM compared with diluents, which contained no milk (TALP, LAC, and EmCare). In Experiment III, centrifuging the s...
Love CC, Blanchard TL, Varner DD, Brinsko SP, Voge J, Bliss S, Sudderth K, Teague S, LaCaze K.An experiment was conducted to determine whether cooled semen quality could be maintained for a longer interval by conducting daily centrifugation of extended semen, with resuspension of the sperm pellet in fresh extender. Semen treatments included SP10NC and SP50NC which contained 10 and 50% seminal plasma, respectively, were not centrifuged (NC), and were stored at 4 to 7 °C for 96 h. Treatments SP10C and SP50C contained 10 and 50% seminal plasma, respectively, but were centrifuged (C) after 24, 48, and 72 h of cooled storage, with daily resuspension in fresh extender containing 10% seminal...
Morillo Rodriguez A, Balao da Silva C, Macías-García B, Gallardo Bolaños JM, Tapia JA, Aparicio IM, Ortega-Ferrusola C, Peña FJ.A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL-1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL-2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 we...
Macías García B, Ortega Ferrusola C, Aparicio IM, Miró-Morán A, Morillo Rodriguez A, Gallardo Bolaños JM, González Fernández L....Glycerol is, to date, the most widely used cryoprotectant to freeze stallion spermatozoa at concentrations between 2% and 5%. Cryoprotectant toxicity has been claimed to be the single most limiting factor for the success of cryopreservation. In order to evaluate the toxic effects of the concentrations of glycerol used in practice, stallion spermatozoa were incubated in Biggers Whitten and Whittingham (BWW) media supplemented with 0%, 0.5%, 1.5%, 2.5%, 3.5%, and 5% glycerol. In two additional experiments, a hyposmotic (75 mOsm/kg) and a hyperosmotic (900 mOsm/kg) control media were included. Sp...
Gallardo Bolaños JM, Miró Morán Á, Balao da Silva CM, Morillo Rodríguez A, Plaza Dávila M, Aparicio IM, Tapia JA, Ortega Ferrusola C, Peña FJ.Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (C...
Ruiz L, Echegaray A, Lafuente A.The aim of this study was the optimization of the sperm freezing protocols for the Pure Breed Andalusian Horse (AH) stallions. The study was performed in 84 ejaculates from 14 stallions (6 ejaculates per stallion). We examined the effect of individual stallion, centrifugal force and centrifugation extender on post-thaw sperm quality. Neither centrifugal force nor centrifugal extender had any significant effect on post-centrifugation or post-thawing sperm quality. Stallion was the principal source of variation in our experiments, showing individual significant differences (p < 0.05) in all para...
Bliss SB, Voge JL, Hayden SS, Teague SR, Brinsko SP, Love CC, Blanchard TL, Varner DD.The objective was to determine if decreased cushion-fluid volume and increased sperm number during centrifugation, or if sperm concentration of extended semen following centrifugation, affected stallion sperm quality. Three ejaculates from each of three stallions were subjected to cushioned centrifugation (1,000g for 20 min). Cushion-fluid volume was set at 1 or 3.5 ml, and sperm number per centrifuge tube was set 1 billion or 3 billion. Following centrifugation, sperm pellets were resuspended in semen extender containing 20% seminal plasma (v/v) with sperm concentrations of 25 or 250 million/...
Hoogewijs MK, de Vliegher SP, Govaere JL, de Schauwer C, de Kruif A, van Soom A.Sperm motility is considered to be one of the key features of semen analysis. Assessment of motility is frequently performed using computer-assisted sperm analysis (CASA). Nevertheless, no uniform standards are present to analyse a semen sample using CASA. Objective: We hypothesised that the type of counting chamber used might influence the results of analysis and aimed to study the effect of chamber type on estimated concentration and motility of an equine semen sample assessed using CASA. Methods: Commonly used disposable Leja chambers of different depths were compared with disposable and re...
Hoogewijs M, Morrell J, Van Soom A, Govaere J, Johannisson A, Piepers S, De Schauwer C, De Kruif A, De Vliegher S.The increasing use of modern reproductive techniques in human medicine has led to a higher demand for isolation of motile sperm. Several of these isolation techniques have been adapted for veterinary use and can be applied for the selection of a superior sperm sample from stallion semen. Until recently a major disadvantage of such isolation techniques was the limitation in sperm volume that could be handled. Androcoll-E had been shown to be successful for processing large volumes of equine semen but there are few data to substantiate the potential beneficial effect of freezing an Androcoll-E s...
Klein C, Troedsson MH.Maternal recognition of pregnancy in the horse is the sum of events leading to maintenance of pregnancy; in a narrow sense, maternal recognition of pregnancy refers to the physiological process by which the lifespan of the corpus luteum is prolonged. The horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. The presence of the conceptus reduces pulsatile prostaglandin F(2α) secretion by the endometrium during early gestation in the mare, partly attributed to the reduced expression of cyclooxygenase-2. Cyclooxygenase-2 has...
Edmond AJ, Brinsko SP, Love CC, Blanchard TL, Teague SR, Varner DD.Centrifugal fractionation of semen is commonly done to improve quality of human semen in assisted-reproduction laboratories, allowing sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, promoting their retention above defined density media, with structurally normal sperm passing through the media following centrifugation. Three experiments were conducted to evaluate the effects of density-medium type, centrifuge-tube size, sperm number, and density-medium volume (column height) on stallion sperm quality and recovery rate in sperm pelle...
Harland MM, Stewart AJ, Böse R.A comprehensive purchase examination is expected by American clients intent on importing a horse from a foreign country. American veterinarians may be involved in performing purchase examinations in foreign countries or, more often, interpreting findings from foreign veterinarians for their American clients. Exportation and importation requirements for horses and semen vary from country to country. Detailed knowledge of the requirements by all involved veterinarians is essential for efficient and successful international equine travel.
Morillo-Rodríguez A, Macías-García B, Tapia JA, Ortega-Ferrusola C, Peña FJ.Ejaculates from six pure Spanish stallions were split, and one subsample frozen in a commercial extender supplemented with the lipid soluble antioxidant butylated hydroxytoluene (BHT), while the other subsample served as control. After at least 4 weeks of storage, samples were thawed and post-thaw sperm quality analysed: sperm motility and kinematics using a CASA system, membrane and acrosome integrity and mitochondrial membrane potential using flow cytometry. The outcome of cryopreservation varied significantly among stallions. However, the supplementation with 1 mm BHT had no significant eff...
Clulow JR, Buss H, Evans G, Sieme H, Rath D, Morris LH, Maxwell WM.Sex-sorted, frozen-thawed stallion spermatozoa remain out of reach of commercial horse breeders because of the low efficiency of the sex-sorting process and unacceptable fertility rates after insemination. Two experiments were designed to test the effects of alternative staining and freezing media to improve the viability of sex-sorted frozen-thawed stallion spermatozoa. Experiment 1 compared two freezing media, INRA 82(®) and a modified lactose-ethylenediaminetetraacetic acid (EDTA), for the cryopreservation of sex-sorted stallion spermatozoa. No significant differences between the two freez...
Papa FO, Felício GB, Melo-Oña CM, Alvarenga MA, De Vita B, Trinque C, Puoli-Filho JN, Dell'Aqua JA.The objective of this study was to determine whether replacing the egg yolk with soybean lecithin in the Botu-Crio® cryodiluent would maintain the fertility of cryopreserved stallion sperm. Two experiments were performed to evaluate cell freezability. In experiment 1, sperm from 15 stallions were frozen in Botu-Crio® (BC) or Botu-Crio® which contained 45g/L soybean lecithin (BCLS45) in place of the egg yolk. In experiment 2, we compared different concentrations of soybean lecithin: 0, 10.0, 12.5, 15.0, 17.5 and 20.0g/L (BC, BCLS10, BCLS12.5, BCLS17.5 and BCLS20, respectively). In experiment...
Pillet E, Labbe C, Batellier F, Duchamp G, Beaumal V, Anton M, Desherces S, Schmitt E, Magistrini M.Egg yolk is normally used as a protective agent to freeze semen of equine and other species. However, addition of egg yolk in extenders is not without disadvantages and the demand to find cryoprotective alternatives is strong. The objective of this study was to test the cryoprotective capacities of liposomes composed of egg yolk phospholipids. Two experiments were conducted: 1) the first to determine the optimal composition and concentration of liposomes to preserve post-thaw motility and membrane integrity of spermatozoa; 2) the second to assess in vivo the cryoprotective capacities of these ...
Monteiro GA, Papa FO, Zahn FS, Dellaqua JA, Melo CM, Maziero RR, Avanzi BR, Alvarenga MA, Guasti PN.The cryopreservation of epididymal sperm is important to preserve genetic material from valuable deceased males. This study evaluated the viability of sperm samples from eight stallions under three conditions: (1) collected using an artificial vagina (EJ-0h), (2) recovered from the epididymal cauda immediately after orchiectomy (EP-0h), and (3) recovered from the epididymal cauda after 24h of storage at 5°C (EP-24h). To obtain EJ-0h sperm, two ejaculates were collected from each stallion. After 1 week, the stallions were submitted to bilateral orchiectomy, and one of the removed epididymides ...
Peña FJ, García BM, Samper JC, Aparicio IM, Tapia JA, Ferrusola CO.We review recent developments in the technology of freezing stallion sperm, paying special attention to the molecular lesions that spermatozoa suffer during freezing and thawing, such as osmotic stress, oxidative damage, and apoptotic changes. We also discuss the applicability of colloidal centrifugation in stallion sperm cryobiology. Increased knowledge about the molecular injuries that occur during cryopreservation may lead to improved protective techniques and thus to further improvements in fertility in the current decade.
Cuervo-Arango J, Aguilar J, Newcombe JR.Embryonic vesicle growth in the mare is easily monitored by ultrasound. Apart from pregnancy diagnosis, assessment of the embryonic vesicle in practice is also useful to evaluate its viability. Although subject to individual variation, embryo growth rate follows a constant pattern in the early stages of development in relation to embryonic age. Previous studies have shown a significant effect of some factors routinely used in practice, such as post-ovulation insemination and embryo transfer, on embryonic growth and the time in which the vesicle is first detected. This study attempts to confirm...
Pommer AC, Linfor JJ, Meyers SA.Preserved stallion semen often has decreased spermatozoal motility and fertility that can vary significantly between individual stallions. It is not known whether the medium used for extending equine sperm contributes to these decreases by inducing premature capacitation during storage. If spermatozoa undergo capacitation or acrosome reaction prior to insemination, this could result in a diminished capacity to penetrate the cumulus mass and fertilize the egg. We hypothesized that skim milk-based semen extenders, similar to those used in cooled storage, stabilize sperm membranes and prolong spe...
Jhamb D, Talluri TR, Sharma S, Juneja R, Nirwan SS, Yadav D, Pargi KK, Tanwar A, Kumar P, Kumar R, Mehta SC, Parashar M, Gaur M.Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. One of the reason for this diminished quality is osmotic stress that spermatozoa experiences during freezing and thawing process. The aim of the present study was to evaluate the cryoprotective effect of trehalose on stallion sperm quality and field fertility rates subjected to cooling and freeze-thawing process. Semen samples were collected from six Marwari breed stallions, divided into three different treatments in a final concentration of 150 × 106 sperm/mL by using Lactose based extender containing...
Deichsel K, Pasing S, Erber R, Ille N, Palme R, Aurich J, Aurich C.The use of breeding stallions for equestrian competitions requires that fertility is not negatively affected by competition or transport to the competition site. In this study, effects of cortisol release induced by road transport (600 km), adrenocorticotropic hormone (ACTH) administration (3 × 0.5 mg synthetic ACTH) and placebo treatment on semen quality and testosterone release were investigated in Shetland stallions (N = 13) using a crossover design. Saliva for cortisol and blood for testosterone analysis were collected for 10 weeks after treatments. Semen was collected daily for 5 days di...
Ramírez-Agámez L, Hernández-Avilés C, Varner DD, Love CC.Intracytoplasmic Sperm Injection (ICSI) using frozen/thawed sperm is a common procedure to obtain embryos from fertile or subfertile mares and stallions. Stallion-associated factors that impact the efficiency of ICSI have been studied less than those associated with the mare. Three experiments were conducted: Experiment 1: the effect of freezing extender composition and cryoprotectant; Experiment 2: the effect of sperm exposure to seminal plasma prior to freezing (ejaculated vs. epididymal sperm; two-freeze/thaw cycles each); and Experiment 3: the effect of sperm morphologic feature used for f...
Olğaç KT, Akçay E.In this study, the effects of polyamines, Spermine and Spermidine, on long-term preservation and post-thaw spermatological parameters were evaluated. Moreover, determination of the most suitable polyamine and its dose that can be added to standard extenders were aimed. Four adult Arabian stallions were used in the study. Five ejaculates were collected from each of four stallions via artificial vagina two days interval. Each ejaculate was divided into 13 aliquots. INRA96 (95,5%), egg yolk (2%), and glycerol (2,5%) were used as a control extender. Extenders of experimental groups were prepared w...
Card C.Morphologic assessment of spermatozoa is one of the most objective measures in a Breeding Soundness Examination of a stallion. There are different systems for morphologic assessment of spermatozoa. The objectives of this article are to review spermatogenesis, describe clinical sample preparation, discuss previous methods of morphologic classification and explain the use of a differential spermiogram. The advantages of the differential spermiogram method of analysis are discussed, along with its use in delineating intrinsic and extrinsic disturbances in spermatogenesis. Case examples of specifi...
Nogueira BG, Pereira RR, Bitencourt JL, Milan B, Reis WVA, Junior MV, Acácio BR, Sampaio BFB, I L Souza M.The objective of the experiment was to evaluate the effect of the addition of different concentrations of the antioxidants Coenzyme Q10 (CoQ10) and melatonin to equine semen freezing diluent, alone or in combination, during the cryopreservation process. Twenty ejaculates (n = 5 stallions) were divided in groups: Control (C, without the addition of antioxidants), melatonin 0.75 mM (MEL1), melatonin 1.5 mM (MEL2), CoQ10 40 μg/mL (Q1), CoQ10 200 μg/mL (Q2), and CoQ10 40 μg/mL+ 0.75 mM melatonin (Q1 +MEL1). Q1 and Q2 groups demonstrated intact plasma membrane and high mitochondrial me...
Kútvölgyi G, Stefler J, Kovács A.A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs....
Rocha CC, Kawai GKV, de Agostini Losano JD, Angrimani DSR, Rui BR, de Cássia Bicudo L, da Silva BDCS, Alonso MA, Mendes CM....Semen biotechniques may impair sperm quality due to excessive production of reactive oxygen species (ROS). Additionally, products of the oxidative reaction, especially involving lipids (e.g., malondialdehyde - MDA), may be even more harmful to sperm. Carnosine, previously reported to be present in seminal plasma of several species, may be a key factor on sperm tolerance to biotechniques by counterattacking the deleterious influence of MDA. Therefore, the aim of this study was to measure the levels of carnosine present in equine seminal plasma and relate these findings with sperm function and o...
Gamboa S, Machado-Faria M, Ramalho-Santos J.The Puro Sangue Lusitano (PSL) is the major national breed of horse in Portugal, but no studies exist on its seminal characteristics, or on the possibility of conserving semen for future use. The aim of this study was to evaluate semen parameters, fertility and the aptness to semen preservation in Lusitano Stallions. In order to compare characteristics defined by a single or by multiple semen collections per stallion 152 ejaculates obtained from 152 Lusitano stallions presented at an annual breeding soundness examination as well as data related to 371 ejaculates obtained from 9 PSL were analyz...
Stawicki RJ, McDonnell SM, Giguère S, Turner RM.The cryopreservation of epididymal sperm can be useful in a variety of circumstances for ensuring genetic preservation of a valued stallion. Although early studies have reported pregnancy rates significantly lower than those achieved with cryopreserved ejaculated sperm, two recent studies report over 60% one-cycle pregnancy rates with epididymal sperm stored for 24 hours at 5 °C before harvest and cryopreservation. The aims of this study were to: (1) attempt to replicate the one-cycle pregnancy rate of over 60% using epididymal sperm cooled and stored within the epididymis for 24 hours before...
Hartmann C, Gerner W, Walter I, Saalmüller A, Aurich C.In the mare, early pregnancy loss is common, but involvement of the maternal immune system in the pathogenesis of this condition has not been investigated in detail so far. In the present study, we assessed effects of exposure of the endometrium to semen or seminal plasma in oestrous mares on the response of regulatory T lymphocytes (Tregs) in the peripheral circulation as well as in the endometrium. Raw semen, seminal plasma or PBS (control) were introduced into the uterus of oestrous mares (n = 12). Blood was collected immediately before insemination or PBS infusion (time 0), and 12, 24 ...
Diaz-Jimenez M, Rota A, Dorado J, Consuegra C, Pereira B, Camillo F, Panzani D, Fanelli D, Tesi M, Monaco D, Hidalgo M.Sperm vitrification has been recently developed, but fertility trials have not been performed yet in equine species. In this study, a new warming technique for vitrified donkey semen was developed and the uterine inflammatory response and fertility were compared to conventional freezing. In Experiment 1, sperm was vitrified in straws and warmed in 3 ml of extender or in a water bath at: 37 °C/30 s; 43 °C/10 s; and 60 °C/5 s. Sperm motility, plasma and acrosome membranes and DNA integrity were compared between treatments. In Experiment 2, jennies were inseminated twice (500 ...
Maziero RRD, Guaitolini CRF, Guasti PN, Monteiro GA, Martin I, Silva JPMD, Crespilho AM, Papa FO.Studies involving different methods and techniques of cryopreservation and its interactions with the conception rates in artificial insemination (AI) programs are reported in the literature. This study evaluated the sperm kinetics, plasma membrane integrity, and fertility rates of mares inseminated with cryopreserved stallion semen subjected to different freezing methods. For this, four ejaculates from five stallions were collected and frozen in conventional (Styrofoam box) or automated system in Mini-Digitcool ZH 400. Seminal samples were evaluated after thawing for sperm motion parameters by...
Heitland AV, Jasko DJ, Squires EL, Graham JK, Pickett BW, Hamilton C.Five experiments were conducted to evaluate damage incurred in each processing step for cryopreservation of stallion spermatozoa. In Experiment 1, semen was centrifuged for 9 centrifugation times and the percentage of spermatozoa recovered after each treatment was calculated and spermatozoal motion characteristics analysed. Recovery of spermatozoa was > or = 80% when spermatozoa were centrifuged for > or = 10 min. Experiment 2 evaluated spermatozoa cryopreserved at 5 different concentrations in each of 2 extenders (skim milk-egg yolk-glycerol, SM-EYG; and lactose-EDTA, LAC). In SM-EYG, T...
Krakowski L, Obara J, Wąchocka A, Piech T, Bartoszek P, Kostro K, Tatara MR.The objective of the study was to assess apoptosis and DNA defragmentation in equine semen diluted and chilled to +4°C. Semen was collected from nine fertile stallions, including four Arabian thoroughbreds and five coldbloods. Examinations were carried out immediately after semen collection (0) and at five storage times (24, 48, 72, 96 and 120 h). The basic semen evaluation was performed in terms of volume, sperm concentration, viable sperm percentage, progressive motility and morphology. Using flow cytometry, DNA defragmentation and cell membrane integrity of spermatozoa were determined. Th...
Ponthier J, Rigaux G, Parrilla-Hernandez S, Egyptien S, Gatez C, Carrasco Leroy C, Deleuze S.This report describes an option to modulate the testicular function of wild horses and field methods to assess it. Non-surgical castration of a captive wild Przewalski's stallion with anti-gonadotropin-releasing hormone (GnRH) immunization was performed by sub-cutaneous injection of two doses of 450 μg (3 ml) of GnRH conjugated to diphtheria toxin, further repeated every 6 months. Semen quality was assessed after collection by electro-ejaculation under general anesthesia. Endocrine and behavioral consequences were studied during a 2-year follow-up period. The procedure of electro-ejaculation ...
Gaitskell-Phillips G, Martín-Cano FE, da Silva-Álvarez E, Tapia JA, Silva A, Gil MC, Ortega-Ferrusola C, Peña FJ.Although recent research has addressed the impact of cryopreservation on the stallion sperm proteome, studies addressing the stallion sperm phosphoproteome are lacking. In the present study, the data set of proteomes of fresh and cryopreserved spermatozoa were reanalyzed, showing that cryopreservation caused significant changes in the phosphoproteome. The phosphoproteins reduced most significantly by cryopreservation were Ca2+binding tyrosine phosphorylation regulated, protein kinase cAMP-activated catalytic subunit beta (CABYR), mitochondria eating protein (SPATA18), A kinase anchoring protei...
Kumar P, Kumar R, Mehta JS, Chaudhary AK, Ravi SK, Chandra Mehta S, Ansari MM, Legha RA, Tripathi BN, Talluri TR.The present study was designed to study the adverse effects of cryopreservation and evaluation of the cryoprotective effect of reduced glutathione (GSH) and ascorbic acid (AA) supplementation on exotic jack semen in combination or alone. For this, 24 semen samples from four adult and fertile jacks were collected via artificial vagina using an estrus jenny as dummy. After semen collection, the semen was evaluated for various qualitative and quantitative parameters in fresh, cooled, and frozen-thawed semen. The semen pellet was extended with the freezing extender containing either AA (0.9 g/L),...
Vidament M, Ecot P, Noue P, Bourgeois C, Magistrini M, Palmer E.The aims of this study were to evaluate the effects of cooling rate to 4 degrees C and temperature at the time of centrifugation/glycerol-addition (freezing extender: INRA82 + 2% egg yolk + 2.5% glycerol) on postcentrifugation recovery rate, post-thaw motility and per-cycle fertility. When centrifugation/glycerol-addition was performed at 4 degrees C (14 ejaculates), a moderate cooling rate (37 degrees C to 4 degrees C in I h) resulted in higher post-thaw motility (45%) than when using a slow cooling rate (37 degrees C to 4 degrees C in 4 h) (39%; P<0.05). When centrifugation/glycerol-addit...
Sotgia S, Taras A, Zinellu A, Cherchi R, Mangoni AA, Carru C, Bogliolo L.The dependence of a stallion's spermatozoa on oxidative phosphorylation for energy requirements results in an unconventional relationship between reactive oxygen species (ROS) production and fertility. In such a scenario, antioxidant activity must be finely controlled and not affect the essential functions of ROS. Some in vivo evidence suggests that the naturally occurring antioxidant ergothioneine (ERT) interferes with the critical roles of ROS/reactive nitrogen species (RNS) in pro-oxidant states but not in healthy tissues. The measurement of ERT in seminal plasma collected from 14 stallions...
Hernández-Avilés C, Love CC, Serafini R, Ramírez-Agámez L, Kelley DE, de Andino EM, Teague SR, LaCaze KA, Brinsko SP, Varner DD.In this study, the effectiveness of supplementing INRA-96® extender (INRA-Control; original antibiotic formulation: potassium penicillin G = 38 μg/mL; gentamicin sulfate = 105 μg/mL; amphotericin B = 0.315 μg/mL) with amikacin sulfate and potassium penicillin G (AP) was determined. In Exp. 1, two sources of amikacin (INRA-AP-Sigma or INRA-AP-GoldBio) in combination with penicillin G were compared with ticarcillin/clavulanate (INRA-Tim) or no-supplemental antibiotics (INRA-Control) to examine effects on sperm quality and commensal bacterial growth. No differences were detected in s...
Janett F, Burkhardt C, Burger D, Imboden I, Hässig M, Thun R.The objective of this study was to investigate changes of quality and freezability of stallion semen in response to repeated acute treadmill exercise. Ejaculates from 11 stallions were collected, evaluated and frozen weekly during four periods of 4 weeks each defined as before (period 1), during (period 2) and after (periods 3 and 4) intense exercise. In fresh semen the gel-free volume, sperm concentration, motility, normal sperm and sperm with major defects (acrosome defects, nuclear vacuoles, abnormal heads, midpiece defects and proximal droplets) were evaluated. In frozen-thawed semen, moti...
Edmond AJ, Brinsko SP, Love CC, Blanchard TL, Teague SR, Varner DD.Centrifugal fractionation of semen is commonly done to improve quality of human semen in assisted-reproduction laboratories, allowing sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, promoting their retention above defined density media, with structurally normal sperm passing through the media following centrifugation. Three experiments were conducted to evaluate the effects of density-medium type, centrifuge-tube size, sperm number, and density-medium volume (column height) on stallion sperm quality and recovery rate in sperm pelle...
Roasa LM, Choi YH, Love CC, Romo S, Varner DD, Hinrichs K.In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; P<0.001). The type of bovine serum albumin used affected fertilization rates (4% versus 39.6%; P<0.001). To determine if IVF rates wer...
Magistrini M, Lindeberg H, Koskinen E, Beau P, Seguin F.The composition of seminal plasma must be determined to assess the possible roles of sex gland secretions in survival of stallion spermatozoa. In the present study, an automated semen collection device and 1H magnetic resonance spectroscopy were used to analyse and compare the composition of seminal plasma from fractionated and nonfractionated stallion ejaculates. The contribution of each semen component to the ejaculate (sequence of production of component and concentration) was evaluated and its relationship to biophysical parameters was determined. 1H magnetic resonance spectroscopy was use...
Stradaioli G, Sylla L, Zelli R, Verini Supplizi A, Chiodi P, Arduini A, Monaci M.The reproductive characteristics and seminal carnitine and acetylcarnitine content as well as carnitine acetyltransferase activity of young Maremmano stallions (n=25) are reported. The stallions were subjected to semen collection in November and January; in each trial two ejaculates were collected 1h apart. The total motile morphologically normal spermatozoa (TMMNS) and the progressively motile spermatozoa at collection and during storage at +4 degrees C were evaluated. Seminal L-carnitine (LC), acetylcarnitine (AC), pyruvate and lactate were measured using spectrophotometric methods, whereas ...
Gáspárdy A, Renkó E, Somoskői B, Bába A, Cseh S.The objective of this study was to compare the efficiency of artificial insemination (AI) carried out with frozen and fresh, diluted and chilled semen under field conditions. One hundred and twenty-nine mares of different breeds were included in the study. Eighty-one out of the 107 mares inseminated with fresh, chilled semen got pregnant. Seven pregnant mares aborted and 74 foals were born. Out of the 22 mares inseminated with frozen semen, 17 mares got pregnant. Two mares out of the 17 pregnant mares aborted and finally 15 healthy foals were born. No difference was found between the two group...
Ghallab ARM, Abou-Ahmed MM, Fad AM, El-Badry DA, Shahat AM, Moawad AR.Cryopreservation of Arabian stallion semen is important in order to improve the function and fertility of frozen/thawed semen in this breed. Objective: To investigate the effects of centrifugation, type of semen extenders, and type of cryoprotectants on the quality of frozen/thawed Arabian stallion spermatozoa. Methods: Semen samples collected from four adult Arabian stallions (one ejaculate per week for 10 consecutive weeks) were either processed directly without centrifugation (no centrifugation; NC) or subjected to centrifugation on the gel-free fraction. Centrifugation protocols were divid...
