Topic:Semen Preservation
Semen preservation involves the collection, processing, and storage of stallion semen for future use in artificial insemination. This practice enables the extension of genetic material across geographical boundaries and temporal constraints. The preservation process typically includes semen evaluation, dilution with extenders, cooling, and sometimes cryopreservation. Factors such as semen quality, extender composition, and storage conditions influence the success of preservation. This page compiles peer-reviewed research studies and scholarly articles that explore techniques, challenges, and advancements in the field of equine semen preservation, focusing on optimizing fertility outcomes and extending the reproductive lifespan of stallions.
[Preservation of genetic variation in domestic animals using biotechnical methods]. The conservation of endangered breeds as live animals is at present the main national strategy of the government and breeding organizations to maintain genetic diversity. Fourty-three breeds and some old strains of cattle, pig, sheep, goat and horses are currently involved. Cryopreservation and banks for sperm, embryos or DNA are another type of genetic material which could subsequently be used for breeding and production in agriculture. Present semen banks involve 9 endangered cattle breeds and also a small amount of deep-frozen sperm of some endangered sheep and horse breeds. Only 2 embryo b...
[Detection of chlamydiae in animal and human semen using direct immunofluorescence]. Frequency of elementary and reticular chlamydial bodies was investigated by direct immunofluorescence tests in ejaculates collected from 52 men, 60 stallions, 42 bulls, and 66 boars using the kits of Progen Biotechnic GmbH and the microscope Labophot-2 Nikon. At the same time, qualitative semen tests, including ejaculate volume, sperm motility, percentage of live and dead sperms and morphological' analyses (Vĕzník and Svecová, 1992) were done. Repeatability of the findings was checked in a group of nine bulls housed at the institute and sampled at weekly intervals for 3 to 4 months (Tab. 1)...
Effects of phosphatidylserine and cholesterol liposomes on the viability, motility, and acrosomal integrity of stallion spermatozoa prior to and after cryopreservation. Computer-assisted motion analyses (CASA) and flow cytometry were used to evaluate stallion spermatozoa prior to and after cryopreservation. Spermatozoa were pretreated with: (1) Hepes-buffered medium (SHB); (2) phosphatidylserine (PS) liposomes; or (3) liposomes composed of both PS and cholesterol (PSCH) prior to dilution in either SHB or skim milk-egg yolk extender (SMEY). After cooling to 5 degrees C in SHB, PS and PSCH pretreatment (23%). Spermatozoal motion parameters were higher for spermatozoa diluted in SMEY than dilution in SHB. In Experiment 2, motion parameters were compared for sper...
Cryopreservation of stallion spermatozoa. The main advantage to using frozen semen in any breeding program is faster genetic gain for the inherited trait desired. Milk production of dairy cows doubled (from 26,000 to 52,000 kg of milk/cow per year) between 1950 and 1980, because the dairy industry was using semen only from bulls with the greatest genetic potential for milk production. This genetic gain could have been achieved without the use of frozen semen; however, the time required to achieve that same genetic progress would have been lengthened exceedingly. Fertility rates using frozen stallion spermatozoa are not equal to that o...
In vitro maturation and fertilization of equine oocytes recovered during the breeding season. The aim of this study was to develope an efficient and reproducible procedure for in vitro maturation (IVM) and fertilization (IVF) in the horse. Cumulus-oocyte complexes (COCs) recovered from the ovaries of mares slaughtered during the breeding season were morphologically evaluated, and those showing a compact cumulus and homogeneously appearing cytoplasm were selected for culture. Effects on the maturation of estrous mare serum (EMS) versus estrous cow serum (ECS) as medium supplement were also evaluated (Experiment 1). In Experiment 2, the fertilization of in vitro matured oocytes with froz...
Effect of timing of frozen semen insemination on pregnancy rate in mares. Thirty-four mares were inseminated with frozen semen from one stallion during 2 oestrous cycles, every 48 h until ovulation took place and within 12 h after ovulation. Semen was frozen using the Colorado method. The insemination dose was from 200 to 400 x 10(6) progressively motile spermatozoa. Ovaries were examined every 12 h to determine time of ovulation. Examination for pregnancy was carried out using ultrasonography, 15 days after ovulation. Thirty-five per cent of mares inseminated < 24 h and 23% of mares inseminated between 24-48 h before ovulation were pregnant (p = 0.388). The pregnan...
Factors affecting motion characteristics of frozen-thawed stallion spermatozoa. 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...
Use of imipramine hydrochloride for treatment of urospermia in a stallion with a dysfunctional bladder. An 8-year-old stallion was evaluated because of recurrent urinary tract infections and chronic intermittent urospermia. After extensive diagnostic testing, it was hypothesized that the stallion had a reflex dyssynergia of the bladder and urethral sphincter. Initial attempts to manage the urospermia included semen fractionation, semen collection after voluntary urination, and use of semen extenders. None of these efforts reliably yielded a quality ejaculate. Administration of imipramine hydrochloride (1.2 mg/kg of body weight, PO, 4 hours prior to semen collection) was initiated in an attempt t...
Effect of various extenders and taurine on survival of stallion sperm cooled to 5 degrees C. Stallion semen was diluted in five different extenders (dimitro-poulus onze (Dimitro's), Kenney's modified tryode (Kenney's), modified INRA82 (INRA82), egg yolk-citrate-taurine (Citrate) and EZ-Mixin) and evaluated for motility after cooling and storage at 5 degrees C for 0, 24, 48, 72 and 96 h. EZ-Mixin extender was used as control while 70 and 100 mM of taurine were added to Dimitro's, Kenney's and INRA82 to study its effect under conditions of storage at 5 degrees C and varying processing modifications. Motility in INRA82 was 57.0, 58.4, 61.1, and 56.1% after 24, 48, 72 and 96 h, respective...
Cryopreservation reduces the ability of equine spermatozoa to attach to oviductal epithelial cells and zonae pellucidae in vitro. Two bioassays were used to evaluate the interaction of fresh and cryopreserved equine semen with oviductal epithelial cells (OEC) and with the zona pellucida (ZP). Split ejaculates were either stored at room temperature or frozen and thawed. In experiment 1, progressive motility and membrane integrity were evaluated for each treatment. Fluorescent labeled spermatozoa were cocultured with monolayers of OEC for 30 minutes, and the number of sperm attached to OEC was counted by fluorescence microscopy and analysis of digitized images. Motility of spermatozoa attached to OEC was observed at 0.5, 3...
Effect of different protein supplements on motility and plasma membrane integrity of frozen-thawed stallion spermatozoa. Three experiments were conducted to evaluate the effect of different macromolecule components (egg yolk, skim milk, and BSA) in a widely employed extender for cryopreservation of horse semen. Spermatozoal motility (MOT) and the percentage of spermatozoa with an intact plasma membrane (IPM) were evaluated in frozen-thawed samples. In the first experiment (four Draft Horse stallions, four ejaculates each) a standard freezing extender containing 20% whole egg yolk was modified by replacing extender components (glucose-EDTA solution, 11% lactose solution) with an increasing volume of a skim milk d...
Use of pharmacologically induced ejaculation to obtain semen from a stallion with a fractured radius. Ejaculation was pharmacologically induced in a 13-year-old Quarter Horse stallion with a spiral fracture of the radius. The owners desired to have semen from the stallion frozen prior to euthanatizing the horse, but because of the debilitating injury, standard methods of semen collection could not be used. With the stallion standing quietly in a stall, a plastic collection bag was positioned over the stallion's penis, and clomipramine hydrochloride (2.2 mg/kg of body weight, IV) was administered. Fifty-five minutes later, xylazine hydrochloride (0.5 mg/kg, IV) was administered. The stallion ej...
Relationship among seminal characteristics, fertility and suitability for semen preservation in draft stallions. Seminal characteristics, fertility and the response to semen preservation (liquid storage and cryopreservation) were evaluated in 4 Draft stallions (Percheron 2, Breton 2). Seminal characteristics (gel-free volume, sperm concentration, sperm morphology, percentage of motile spermatozoa) were assessed in 5 ejaculates from each of the 4 stallions. The fertility of the stallions was calculated retrospectively as the accumulated pregnancy rate over 3 breeding seasons. Five ejaculates from each of the stallions were subjected to liquid storage at 5 degrees C. The percentage of motile spermatozoa (P...
Recent developments in cryopreservation of stallion semen with special emphasis on thawing procedure using thermal hysteresis proteins. This research study explores the process of cryopreservation of stallion semen, focusing on improving the thawing procedures using thermal hysteresis proteins (THPs) from Antarctic and Arctic fish in order to […]
Effect of seminal plasma on motion characteristics of epididymal and ejaculated stallion spermatozoa during storage at 5 degrees C. The objective of this experiment was to examine the effect of seminal plasma on motion characteristics of epididymal and ejaculated equine spermatozoa during storage at 5 degrees C. Epididymal spermatozoa were flushed with either seminal plasma or a skim milk-glucose extender. Ejaculated spermatozoa were collected with extender added 10 minutes after semen collection and addition of extender during ejaculation by placing 50 ml extender in the collection bottle. Semen samples were centrifuged and resuspended with a skim milk-glucose extender containing seminal plasma (0, 5 and 25%; v/v), prepar...
Sperm-induced leukocytosis in the equine uterus. The objective of this study was to investigate the inflammatory reaction induced in the equine uterus by insemination with fresh and frozen semen. Eleven groups (6 to 8 mares per group) were studied during 2 breeding seasons. The mares were inseminated using raw semen, frozen semen, extended fresh and frozen semen, concentrated fresh semen, seminal plasma and seminal extenders only. One group was bred naturally. Six hours after insemination, the uteri were flushed with 50 ml of phosphate-buffered saline (PBS). Seventeen out of 104 samples (16%) exhibited slight bacterial growth. Neutrophil con...
Insemination results with slow-cooled stallion semen stored for approximately 40 hours. Semen from 3 stallions was extended using 2 methods (Kenney extender and a modified Kenney extender), slowly cooled, and stored for 41 +/- 6 (s.d.) h before insemination. An insemination dose (40 ml) contained 1.5-2 billion spermatozoa. In the experiment, 26 mares were inseminated in 30 cycles. The pregnancy rate per cycle obtained with sperm stored in the Kenney extender was 87% (n = 15). When the semen was extended with the modified extender, centrifuged and stored, the pregnancy rate was 60% (n = 15). Inseminations were done every other day until ovulation was detected. If a mare ovulated m...
Post-thaw motility and longevity of motility of imipramine-induced ejaculates of pony stallions. Imipramine-induced ex copula ejaculates (11) and fractionated in copula ejaculates were collected from each of 5 pony stallions for freezing in 5-ml straws (6), using a modified Kenney glucose skim-milk extender (2). Initial post-thaw total and progressive motilities and daily post-thaw total and progressive motilities, as well as the number of days to reach 0 progressively motile spermatozoa, were also similar for the 2 methods of collection. The percentage of morphologically normal spermatozoa both before freezing and after thawing were also similar for in copula and ex copula ejaculates. Co...
Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing. The suitability of ejaculated and epididymal stallion spermatozoa for cooled storage (5 degrees C) and cryopreservation was examined in 5 ejaculates from each of 6 stallions and in spermatozoa recovered from the cauda epididymidis after castration of these stallions. The percentage of progressively motile spermatozoa, examined by subjective estimation (cooled samples) or by computerized analysis (frozen-thawed samples), was used as parameter. In ejaculated semen samples containing 5 and 25% seminal plasma in a skim milk glucose extender, the lower amount of seminal plasma supported spermatozoa...
Freezability and fertility results with uncentrifuged stallion semen. The first (1 to 3) sperm-rich fractions of the ejaculate were collected from 4 stallions using an open-ended vagina. The volume of the collected fractions was 12 +/- 8 ml with a density of 475 +/- 200 million spermatozoa/ml. Before freezing, the semen was diluted with a skim-milk based extender 1:1 to 1:8 (volume of semen: volume of extender), depending on the initial sperm concentration to achieve a final concentration of 100 million/ml. The total number of spermatozoa in an insemination dose ranged from 0.7 to 1 billion spermatozoa. Within 12 h after ovulation, 48 mares were inseminated in 7...
[Cryopreservation trial with semen of purebred Arabian and Haflinger stallions in the Turkish national stud in Karacabey]. Within a German-Turkish university partnership deep freezing preservation of stallion semen was performed as a part project of the cooperation contract. In this study a modification of the introduced Makrotüb method was used for semen freezing. The investigated characteristics of fresh semen of the Arab stallions were in the normal range cited in the international literature. However, the semen data obtained from the Haflinger stallions were markedly and partially significantly in lower range than measured for the Arab stallions. This may reflect an incomplete adaptation process of the import...
The effectiveness of gentamicin or polymyxin B for the control of bacterial growth in equine semen stored at 20 degrees C or 5 degrees C for up to forty-eight hours. Semen from three stallions was used to evaluate the effectiveness of two antibiotics added to semen extender for samples stored at 20 degrees C or 5 degrees C for up to 48 hours. Each ejaculate was divided into six different treatments: semen+extender (SE); SE+gentamicin (100 micrograms/mL); SE+polymyxin B (1000 units/mL); and each of the above treatments inoculated with Pseudomonas aeruginosa ATCC 27853. Sampling of diluted semen for bacteriological analysis was performed after 2, 8, 24 and 48 hours of preservation at either temperatures. The presence of nonspecific bacteria was noted after t...
Capacitation-like membrane changes and prolonged viability in vitro of equine spermatozoa cultured with uterine tube epithelial cells. Reliable capacitation of equine spermatozoa has been a major obstacle in the development of equine in vitro fertilization. Experiments were done to compare in vitro capacitation of equine spermatozoa by use of heparin/caffeine, calcium ionophore, uterine tube epithelial cell (UTEC)-conditioned medium, and direct culturing of spermatozoa with UTEC (coculturing). Capacitation-like changes, as determined by chlortetracycline membrane staining patterns, developed with UTEC-conditioned medium and coculturing, equivalent to that with calcium ionophore. Both of these treatments induced more (P < 0.05...
Liquid storage and freezing of semen from New Forest and Welsh Pony stallions. Two experiments were conducted to examine the effects of liquid storage extender and of a modified freezing protocol on motility and morphology parameters of 3-year-old pony stallions. In experiment 1 ejaculates were diluted 1 + 1 (v+v) with glycine-egg-yolk extender (D11) or skim milk extender (SME), centrifuged, resuspended in the corresponding extender and kept at +5 degrees C. Concerning motion characteristics, both progressive motility and average path velocity of semen stored in SME was significantly superior to semen stored in D11 after 6, 18 and 42 hrs. However, over time of storage th...
Column separation of motile sperm from stallion semen. Subfertility in stallions is common, and methodologies are needed to increase the fertility in these animals. In other species, removal of the dead sperm from semen increases the quality and fertility of semen. With horse semen we evaluated 48 combinations of column separation techniques using micro-spin chromatography columns. The greatest improvement in motility was observed with glass wool, whereas glass beads exhibited the greatest recovery of motile sperm. Although centrifugation time did not influence recovery rate or percent motility, a column length of 2 cm was superior for recovery of...
Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review. When sperm of the ram, bull, boar and stallion are cold-shocked by rapid cooling to near freezing point, motility and metabolic activity are irreversibly depressed and the acrosome and plasma membrane disrupted. Ram sperm become susceptible to cold shock in the proximal corpus region of the epididymis when the cytoplasmic droplet has moved backwards to the distal portion of the sperm midpiece. The membrane constituents phospholipids and cholesterol are important in cold shock which causes loss of lipid from sperm. The susceptibility of sperm to cold shock is linked with a high ratio of unsatur...
Semen collection techniques. Semen collection techniques in the stallion have evolved considerably over the last 70 to 80 years and are used today primarily for artificial insemination. Semen can be collected from stallions that are otherwise unable to breed, allowing continued use of valuable animals. There are many options for collection of semen from stallions that present with ejaculatory dysfunction (see the article by McDonnell elsewhere in this issue.) Although there are many advantages to the use of artificial breeding, the collector must understand each step of the collection procedure as well as stallion prefere...
Artificial insemination and preservation of semen. Artificial insemination is an effective technique for improving utilization of stallions in breeding programs. When proper semen handling and insemination procedures are used, optimal pregnancy rates are attainable. When AI techniques are employed for mares and stallions with marginal fertility, pregnancy rates may be improved in comparison with natural mating. Preservation of stallion semen in the liquid or frozen state reduces the costs and potential health hazards incurred by transporting mares and provides easier access to genetic material that may otherwise be unavailable. Acceptable preg...