Topic:Cryopreservation
Cryopreservation in horses involves the process of cooling and storing biological samples at very low temperatures to preserve their viability for future use. This technique is primarily applied to equine gametes, embryos, and genetic material, such as semen and oocytes. The process aims to halt all biological activity, thereby maintaining the integrity of the samples over extended periods. Cryopreservation is utilized in equine breeding programs to enhance genetic diversity, support conservation efforts, and facilitate international genetic exchange. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, challenges, and applications of cryopreservation in equine reproduction and genetics.
Significance of plasmalemma disruption in bovine and equine spermatozoa. We have investigated fresh and cryopreserved bovine and equine spermatozoa using light and transmission soft X-ray microscopy. Spermatozoa were examined, in the presence or absence of semen, after using Percoll gradient centrifugation and re-suspending in medium. X-ray microscopy provided high resolution (30 nm) transmission images of whole cells in solution with high contrast, while retaining the simple preparation techniques used in light microscopy. We demonstrated translucent, membrane-bound vesicles in the acrosomal and midpiece regions that were similar in size and we noted their inciden...
The use of pentoxifylline to improve motility of cryopreserved equine spermatozoa. Pentoxifylline was evaluated as a method to increase motility of cryopreserved equine spermatozoa. In a preliminary experiment, pentoxifylline (3.5 mM or 7.0 mM) was added to extended semen that was chilled to 4 degrees C. Motility was evaluated at 8-h intervals for 48 h. The addition of 3.5 or 7.0 mM pentoxifylline appeared to increase the motility of chilled spermatozoa compared to controls. Based on these results, similar concentrations of pentoxifylline were added to semen either before or after cryopreservation. The addition of pentoxifylline (3.5 or 7.0 mM) to semen before cryopreservati...
Centrifugation and addition of glycerol at 22 degres C instead of 4 degrees C improve post-thaw motility and fertility of stallion spermatozoa. 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...
Acrosomal ultrastructure of stallion spermatozoa cryopreserved with ethylene glycol using two packaging systems. The present experiments aimed to examine the substitution of glycerol (G) by ethylene glycol (E) as a cryoprotective agent for stallion spermatozoa. Two different ethylene glycol concentrations (5% and 10%) and also the association of glycerol (2%) and ethylene glycol (3%) (E/G) were studied (Experiment 1). In Experiment 2, two packing systems (0.5 x 4.0 ml) were evaluated using both cryoprotectors. In both experiments, the sperm membrane integrity after freezing was evaluated using transmission electron microscopy. The mean post-thaw motility was 34.25, 36.5, 29.25 and 34.75% for G5%, E5%, E1...
Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage. The objective of this study was to determine if centrifugation and partial removal of seminal plasma would improve spermatozoal motility in semen from stallions whose whole ejaculates have poor tolerance to cooling and storage. Stallions were divided into two groups (n = 5/group) based on the ability of their extended semen to maintain spermatozoal motility after cooling and storage. Group 1 stallions ("good coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by < or = 30% of progressive motility prior to storage. Group 2 stallions ...
Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws. Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol. Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EFS) for 30 sec and then to 5 M ethylene...
Assessment of equine sperm mitochondrial function using JC-1. The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged thro...
Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa. This study was conducted to compare the cooling rates and storage temperatures within equine semen transport containers exposed to different ambient temperatures, and to evaluate the ability of these containers to preserve spermatozoal motility following 24 h of storage under these conditions. In Experiment 1, nonfat dried milk solids, glucose, sucrose, equine semen extender was divided into seven 40-mL aliquots and loaded into seven different semen transport containers: Equitainer I, Equitainer II, Equitainer III, ExpectaFoal, Bio-Flite, Lane STS, and Equine Express. After containers were loa...
Effects of glutamine, proline, histidine and betaine on post-thaw motility of stallion spermatozoa. The supplementation of the freezing diluent with 3 amino acids (glutamine, proline and histidine) and 1 amino acid-related compound (betaine) in preserving stallion spermatozoa diluted in INRA82 extender containing 2.5% (v/v) glycerol and 2% (v/v) egg yolk (control extender) during freezing and thawing was studied at 0, 40, 80, 120 and 160 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 1). Glutamine and proline were studied at 0, 10, 20, 30, 40, 50, 60, 70 and 80 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 2). In each experiment, spermatozoa were eval...
Sperm transport and survival in the mare: a review. After the deposition of semen in the mare's uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mare's reproductive tract. Fertilizable sperm are present in the oviduct within 4 h after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa...
Current methods for stallion semen cryopreservation: a survey. Various factors affect the success of AI with frozen-thawed semen in horses. Stallion variability is thought to be one of the major factors, but semen processing and evaluation techniques, thawing protocols, packaging systems and timing of insemination are far from standardized among laboratories. Our objective was to survey current methods for stallion semen cryopreservation used commercially around the world. From the answers to the questions in the survey, we attempted to provide an overview of procedures that are standard as well as those that are used by only few laboratories and to revie...
Hypoosmotic test in equine spermatozoa. The aim of the study was to evaluate equine sperm membrane integrity using the hypoosmotic swelling (HOS) test and to correlate this test with different sperm parameters in raw and frozen thawed semen. The HOS solutions were made with fructose, sucrose, lactose and sodium citrate each at 300, 150, 100, 50 and 25 mosm. Maximum numbers of swollen spermatozoa were observed in solutions of fructose, sucrose and lactose each at 100, 50 and 25 mosm. Correlations between progressive motility, morphologically normal spermatozoa and the HOS test were r = 0.75 and r = 0.51 in raw semen and r = 0.26 and ...
Quality of stallion semen obtained by a new semen collection phantom (Equidame) versus a Missouri artificial vagina. A study was performed to test a new semen collection device (Equidame phantom) that fractionates the ejaculate by comparing the quality of semen obtained by the Equidame phantom with that obtained by a Missouri artificial vagina. Semen from 4 Finnhorse stallions was collected 4 times per stallion by both methods. Half of the ejaculate was frozen and the other half extended and loaded into 2 Equitainer transport containers (24- and 48-h samples). Motility parameters were determined by a Hamilton-Thorn motility analyzer after cooled storage for 24 and 48 h and again after freezing/thawing. Raw a...
The current status of equine embryo transfer. The use of embryo transfer in the horse has increased steadily over the past two decades. However, several unique biological features as well as technical problems have limited its widespread use in the horse as compared with that in the cattle industry. Factors that affect embryo recovery include the day of recovery, number of ovulations, age of the donor and the quality of sire's semen. Generally, embryo recoveries are performed 7 or 8 d after ovulation unless the embryos are to be frozen, in which case recovery is performed 6 d after ovulation. Most embryos are recovered from single-ovulati...
In vitro and xenogenous capacitation-like changes of fresh, cooled, and cryopreserved stallion sperm as assessed by a chlortetracycline stain. Like the human female, the mare experiences reproductive tract pathology that may sometimes be circumvented by the use of assisted reproductive technologies (ARTs). One such technology, gamete intrafallopian transfer (GIFT), may be used in mares that exhibit ovulatory, oviductal, or uterine abnormalities that limit the use of common ARTs, such as embryo transfer. Homologous GIFT has been successfully performed in the horse; however, the logistics, costs, and associated risks of surgically transferring gametes to the oviducts of a recipient mare are considerably high. Use of a less costly speci...
Effect of cryopreservation and oviductal cell conditioned media on Ca2+ flux of equine spermatozoa. Movement of Ca2+ into spermatozoa is a critically important event for capacitation and the acrosome reaction. In the present study, the nature of Ca2+ movement in fresh equine spermatozoa was established and the effects of oviductal cell conditioned medium (OCM) and cryopreservation on Ca2+ flux were investigated. The ability of fresh and cryopreserved stallion spermatozoa to regulate Ca2+ concentration over time was evaluated in Ca2+ -free PBS. Intracellular Ca2+ concentrations were higher in cryopreserved spermatozoa than in fresh spermatozoa. However, extracellular Ca2+ concentrations were ...
Effect of cholesterol on the motility and plasma membrane integrity of frozen equine spermatozoa after thawing. The aim of the present study was to investigate the cryoprotectant properties of cholesterol after incorporation into the plasma membranes of equine spermatozoa. A cholesterol-methyl-beta-cyclodextrin complex was used to alter sperm plasma membrane cholesterol content. Ejaculates from six stallions were centrifuged in a non-fat skimmed milk glucose-sucrose extender (MK) or a modified Tyrode's medium (TALP). The sperm pellets were resuspended in the appropriate extender with or without added cholesterol (0.125 mmol cholesterol-methyl-beta-cyclodextrin complex l(-1)) and incubated at 24 degrees ...
Freezing of stallion semen: interactions among cooling treatments, semen extenders and stallions. In the present study, the interactions among stallions, semen extenders and cooling treatments before stallion semen samples were frozen were studied. In Expt 1, the effects of four cooling treatments and three semen extenders were investigated (11 stallions x four split ejaculates), whereas in Expt 2, the effects of two semen extenders, two egg yolk concentrations and two glycerol concentrations were investigated (six stallions x five split ejaculates). Sperm motility after thawing was evaluated. In Expt 1, the extender x cooling treatment interaction was significant. Centrifugation and addit...
Effect of L-glutamine for freezing equine embryos: evaluation by DAPI staining and transfer of multiple embryos to recipient mares. Day 6.5 equine embryos (n=30) were frozen in a medium containing glycerol (2.5-10.0%) supplemented with 0, 20 or 100 mmol L-glutamine 1(-1). After thawing, the embryos were tested individually, using 4',6'-diamidino-2-phenylindole (DAPI) staining to evaluate cell death. Three embryos (one frozen at each L-glutamine concentration) were transferred together into individual recipient mares. Pregnancy diagnosis was performed at day 12 (age of embryo). Embryos were collected at day 14 (age of embryo) and were identified by PCR amplified microsatellite analysis. Nine of ten recipient mares that rece...
Motility, morphology and triple stain analysis of fresh, cooled and frozen-thawed stallion spermatozoa. The aim of the present study was to determine whether there are characteristics of fresh, cooled and frozen-thawed semen samples that can be used to predict the suitability of stallion semen for preservation by cooling or freezing. Each of three ejaculates obtained from 12 stallions was divided into aliquots to be analysed for sperm motility, morphology and membrane integrity as fresh, cooled and frozen-thawed samples. The percentage of morphologically normal spermatozoa was similar in fresh and cooled samples and both were greater than in the frozen samples. There were no strong linear relati...
Comparison of the cryoprotectant properties of glycerol and ethylene glycol for early (day 6) equine embryos. Early (day 6) equine embryos (n=23) were assigned to four treatment groups to assess the cryoprotectant properties of glycerol and ethylene glycol and the effect of adding sucrose during removal of the cryoprotectant: (i) group GG (n=5) embryos were frozen and thawed using 1.5 mol glycerol l(-1) as the cryoprotectant, which was added at 22 degrees C in four steps (0.375, 0.75, 1.125 and 1.5 mol glycerol l(-1)), and removed after thawing in five steps (1.5, 1.125, 0.75, 0.375 and 0.0 mol glycerol l(-1)); (ii) group GS (n=6) embryos were frozen and thawed using 1.5 mol glycerol l(-1) as for grou...
In vitro interactions of cryopreserved stallion spermatozoa and oviduct (uterine tube) epithelial cells or their secretory products. Formation of a spermatozoa ('sperm') reservoir in the mare is thought to occur through lectin-mediated sperm attachment to the oviductal epithelium. Once attached, prefertilization sperm survival is supported by oviductal factors. Cryopreservation of stallion sperm decreases the number of sperm attaching to oviduct epithelial cells (OEC) and the length of time these sperm survive. Quantification of in vitro interactions between sperm and OEC in a co-culture system may provide an assay for functional integrity of cryopreserved or fresh sperm samples. Additionally, superior additives for in vitr...
Freezing of stallion semen with addition of glycine betaine. The effect of addition of glycine betaine to a lactose-EDTA freezing medium on the post-thaw motility of stallion semen was determined. The first three semen-rich fractions of nine stallions were collected with an open-end Krakow artificial vagina on consecutive weekdays. Semen was frozen using the Hannover method with freezing media containing glycine betaine in various concentrations from 0 to 5%. After thawing, sperm motility was analysed both by a light microscope and by a Hamilton-Thorn Motility Analyser. Total and progressive post-thaw motilities of semen containing 0.25-3% glycine betai...
Effects of bovine serum albumin on function of cryopreserved stallion spermatozoa during medium culture and uterine tube epithelial cell coculture. To compare function of cultured cryopreserved stallion spermatozoa in a modified Tyrode's medium (TM), with or without bovine serum albumin (BSA), or in uterine tube (oviduct) epithelial cell (OEC) coculture in TM, with or without BSA. Methods: Cryopreserved spermatozoa from 6 proven stallions and OEC from bovine reproductive tracts in follicular phase. Methods: Thawed spermatozoa were cultured in TM, with or without BSA, or cocultured with OEC monolayers in TM, with or without BSA. Percentages of capacitated and acrosome-reacted spermatozoa were measured at 5 hours for TM cultures. Spermatozo...
Prostasome-like particles in stallion semen. Human semen contains membranous vesicles called prostasomes. They are secreted by the prostate gland and contain large amounts of cholesterol, sphingomyelin, and Ca2+. Prostasomes enhance the motility of ejaculated spermatozoa and are involved in a number of additional biological functions. No prostasome-like vesicles have been described in horse semen up to now. We have demonstrated the presence of prostasome-like vesicles in the equine semen and characterized them as to size, morphology, and lipid composition; we have found that they are similar to human prostasomes in many respects. We prop...
The effect of propanediol on the morphology of fresh and frozen equine embryos. Seventeen horse embryos recovered on the sixth day after spontaneous ovulation were; 1) washed in PBS (n = 6), 2) treated with 1.5 M 1-2 propanediol (n = 6) or, 3) frozen and thawed using 1.5 M propanediol as the cryoprotectant (n = 5). After treatment, the embryos were incubated for 6 h in medium before they were fixed, serially sectioned and examined microscopically to count the total numbers of interphase, mitotic and pycnotic nuclei. Significant differences were measured only in the mean proportions of pycnotic cells (+/- s.d.), both between the control (9.2 +/- 7.3%) and frozen-thawed emb...
The effect of sucrose in the thawing solution on the morphology and mobility of frozen equine embryos. Seventy-five embryos were collected 6 days after ovulation. Sixty embryos were frozen in straws using glycerol as the cryoprotectant in an automatic freezer. In Experiment 1 the freezing and thawing media were supplemented with 1.3 g/l PVP; in Experiment 2 the supplement was 5% FCS. The embryos were thawed for 30 s at +37 degrees C in a waterbath. In Experiment 1 glycerol was removed from 10 embryos in 6 steps. In 10 other embryos, glycerol and sucrose were both removed from the medium in 6 steps. After glycerol and sucrose removal, the embryos were stained with 4',6'-diamidino-2-phenylindole ...
Cryopreservation procedures for Day 7-8 equine embryos. Larger grade 1 or 2 (1 = excellent,.... 4 = degenerate) equine embryos that ranged in diameter from 300 to 680 microm and were recovered from mares on Day 7 or 8 after ovulation, were randomly assigned to 3 widely divergent cryopreservation treatments. Treatment 1 consisted of cooling from -6 degrees C to -35 degrees C at 0.5 degrees C per min followed by plunging into liquid nitrogen, with a one-step addition and a 4-step removal of 1.0 M glycerol. Treatment 2 (step-down equilibration) consisted of a 2-step addition of glycerol to 4.0 M followed by a decrease to 2.0 M prior to freezing, with ...
Cryopreservation of equine embryos with glycerol plus sucrose and glycerol plus 1,2-propanediol. Six or 7-day-old equine embryos were divided into 4 groups; Group 1, n = 15, Day 7 embryos destined for immediate transfer; Group 2, n = 15, Day 6 embryos destined for deep-freezing with glycerol plus sucrose as cryoprotectant; Group 3, n = 10, Day 6 embryos destined for deep-freezing with glycerol plus 1,2-propanediol as cryoprotectant and Group 4, n = 3, fresh embryos destined for ultrastructural analysis. All the frozen/thawed embryos were transferred to recipient mares, except 3 embryos in Group 3 that were subjected to ultrastructural analysis. After thawing the cryoprotectants were remov...
Effect of anti-freeze protein (AFP) on the cooling and freezing of equine embryos as measured by DAPI-staining. Equine embryos recovered on Day 6 after ovulation were cooled to +4 degrees C, or frozen with AFP alone or together with glycerol. Twenty embryos (140-200 microm in diameter) were randomly assigned to 6 treatment groups. In the first 3 groups, the embryos were cooled from room temperature to +4 degrees C at a rate of 3 degrees C/min and warmed again at a rate of 32 degrees C/min in a programmable freezer. In the second 3 groups, the embryos were frozen using a standard protocol, stored in liquid nitrogen for 5-7 days and then thawed in a 37 degrees C waterbath. After cooling/warming or freezin...