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.
Sperm transport and endometrial inflammatory response in mares after artificial insemination with cryopreserved spermatozoa. This study aimed to determine whether the insemination site and dose with cryopreserved sperm of reproductively normal mares affect the sperm population in uterine tubes and the intensity of endometrial inflammatory response. Experimental subjects were estrous mares inseminated, in the mid-uterine body (Body) or the tip of the uterine horn (Tip), ipsilateral to the dominant follicle, with one 0.5 mL straw with 50 × 106 sperm (50) or with eight straws with 50 × 106 sperm/straw (400). Mares were slaughtered 2 h, 4 h and 12 h after artificial insemination (AI) and randomly assigned to f...
Effect of Seminal Plasma Protein Fractions on Stallion Sperm Cryopreservation. Seminal plasma (SP) is the natural environment for spermatozoa and contains a number of components, especially proteins important for successful sperm maturation and fertilization. Nevertheless, in standard frozen stallion insemination doses production, SP is completely removed and is replaced by a semen extender. In the present study, we analyzed the effects of the selected seminal plasma protein groups that might play an important role in reducing the detrimental effects on spermatozoa during the cryopreservation process. SP proteins were separated according to their ability to bind to hepar...
Semen quality and freezability analysis during breeding and non-breeding seasons in heavy draft stallions in southern Chile. The aim of this study was to evaluate seasonal changes in basic parameters of sperm quality and freezability behaviour of ejaculates from 10 fertile heavy draft stallions. A total of 140 ejaculates were collected, processed and evaluated during both the breeding (September-November) and non-breeding seasons (April-June). Fresh semen was evaluated for volume, concentration, total spermatozoa per ejaculate, plasma membrane integrity and total sperm motility. Cryopreserved samples were evaluated for plasma membrane integrity and sperm motility by the CASA system, and for the freezability index (F...
Cholesterol Loaded Cyclodextrin Supplementation Enhances the Cholesterol-to-Phospholipid Ratio and Diminishes Oxidative Stress in Jack Spermatozoa During Cryopreservation. The present study was conducted with the hypothesis that addition of cholesterol to the extender would stabilize the sperm membranes by increasing the cholesterol-to-phospholipid (C:P) ratio and would result in an improved post-thaw semen quality and reduce oxidative stress in the jack (Martina franca) semen. Forty-eight ejaculates from six jacks were collected and analyzed for the present study. The freshly collected semen sample of each jack stallion was divided into five equal fractions after addition of the primary extender without cholesterol-loaded cyclodextrin (CLC) (C) and with 1, 1.5,...
Nicotinic Acid (Niacin) Supplementation in Cooling and Freezing Extenders Enhances Stallion Semen Characteristics. In the present study, we aimed to evaluate the possible protective effects of the nicotinic acid (NA) at three concentrations (10, 20, and 40 mM) on the equine cooled and frozen-thawed spermatozoa quality markers including viability, plasma membrane or acrosome integrity, DNA fragmentation, lipid peroxidation, and total oxidant levels. We also evaluated the effects of NA on preservation of the post-thaw sperm quality after 6 hours of cold storage before freezing. Five stallions were used for semen collections. The current experiment was repeated six times using pooled semen samples from two ...
Optimization of cryopreservation protocols for cooled-transported stallion semen. Freezing cooled-transported semen allows veterinarians and breeders to collect and process the semen of stallions on farm, and then ship the semen to a semen freezing center. There, however, is a lack of standardization of shipping and freezing protocols. The objectives were to optimize and simplify protocols to freeze cooled-shipped semen. In Experiment 1, cooled-transported semen was centrifuged at room temperature or 5 °C before freezing. Sperm variables (motility, membrane integrity, acrosome integrity, membrane fluidity) were evaluated before and after freezing. Centrifugation temperat...
Optimal activation methods for maximizing the concentrations of platelet-derived growth factor-BB and transforming growth factor-β1 in equine platelet-rich plasma. Platelet-rich plasma (PRP) therapy has been widely applied in various medical fields including humans and horses. This study aimed to establish an optimal activation method to stably and reproducibly maximize the concentrations of platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) contained in equine PRP. Autologous PRP was prepared from 11 Thoroughbreds. For the activation test, PRP was activated by either a single freeze-thaw cycle (Fr) or adding calcium and autologous serum containing thrombin (Ca). PDGF-BB and TGF-β1 concentrations in Fr, Ca, nonactiv...
Vitrification of Equine In Vivo-Derived Embryos After Blastocoel Aspiration. Embryo cryopreservation is normally performed with great success in species like humans and cattle. The large size of in vivo-derived equine embryos and the presence of a capsule-impermeable to cryoprotectants-have complicated the use of embryo cryopreservation in equine reproduction. A breakthrough for this technique was obtained when large equine embryos could be successfully cryopreserved after collapsing the blastocoel cavity using a micromanipulation system. High pregnancy rates have been obtained when vitrification is used in combination with embryo collapse.
Cryopreservation of Semen from Domestic Livestock: Bovine, Equine, and Porcine Sperm. In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow cooling from room temperature to 5 °C, packaging of insemina...
Alternative Protocol Using Imipramine, Detomidine, and Oxytocin for Semen Collection in Stallion with Ejaculatory Dysfunction. A 7-year-old Quarter Horse stallion was admitted at the hospital with a history of ejaculatory failure for 12 months. The stallion revealed no physical or psychological abnormalities, as well as, normal libido and erection. In addition, there were no abnormalities in accessory sex glands or the aorta artery detected by transrectal ultrasonography. Based on clinical findings, the stallion was diagnosed with an idiopathic ejaculatory dysfunction; therefore, alternative attempts of semen collection were performed. Thermal compress on the basis of the stallion's penis, semen collection on the gro...
Effect of MnTBAP on in vitro capacitation of frozen-thawed stallion sperm. In vitro manipulation of spermatozoa leads to deleterious changes of structure and function that occur mainly due to oxidative stress, therefore, prevention or treatment is a strategy to improve the functions of processed sperm. In the present study, the aim was to evaluate the effects of MnTBAP supplementation, a compound with antioxidant activity, on in vitro capacitation conditions of thawed equine sperm. For this purpose, stallion spermatozoa (2 × 10 cells/mL) were incubated in the sperm-TLP base medium for 4 h in which there were three different conditions: non-capacitating, capacitating...
Irradiating frozen-thawed stallion sperm with red-light increases their resilience to withstand post-thaw incubation at 38 °C. The aim of this study was to evaluate whether red-light stimulation increases the longevity and resilience of cryopreserved stallion sperm to withstand post-thaw incubation for 120 min. Sixteen frozen straws of 0.5 mL from eight stallions were used. Samples were cryopreserved, thawed through incubation at 38 °C for 30 s and divided into the control and samples exposed to red-light using a triple LED photo-activation system (wavelength: 620-630 nm). Three irradiation protocols consisting of different light-dark-light intervals (1-1-1, 2-2-2 and 3-3-3 min) were tested. Sperm quality param...
Embryo technologies in donkeys (Equus Asinus). In industrialized countries, the donkey population had a dramatic decrease during the last century and this has brought almost all European donkey breeds to risk. Embryo technologies have already been employed as a tool for the conservation of endangered equid species (e.g. Equus Przewalskii). Today it is possible to obtain pregnancies after the transfer of donkey embryos in synchronized recipients, even though embryo transfer is not widely used as a reproductive technique in this species. So far, very few foals are born after transfer of cryopreserved embryos. To date, no pregnancies have bee...
Supportive techniques to investigate in vitro culture and cryopreservation efficiencies of equine ovarian tissue: A review. During the reproductive lifespan of a female, only a limited quantity of oocytes are naturally ovulated; therefore, the mammalian ovary possesses a substantial population of preantral follicles available to be handled and explored in vitro. Hence, the manipulation of preantral follicles enclosed in ovarian tissue aims to recover a considerable population of oocytes of high-value animals for potential application in profitable assisted reproductive technologies (ARTs). For this purpose, the technique of preantral follicle in vitro culture (IVC) has been the most common research tool, achievin...
Effects of cysteine and ascorbic acid in freezing extender on sperm characteristics and level of enzymes in post-thawed stallion semen. Present study aimed to investigate the effect of adding antioxidants, cysteine and ascorbic acid on the levels of glutamic oxaloacetic transaminase (GOT), glutamic-pyruvate (GPT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (GGT) enzymes of post-thawed stallion sperm. Ten ejaculates were collected each from four healthy stallions and cryopreserved using HF-20 freezing extender containing either 0 mg/ml cysteine or ascorbic acid, 0.5 mg/ml cysteine and 0.5 mg/ml ascorbic acid. All samples in freezing extender containing cysteine or ascorbic acid or ...
Post-thawing Sperm Quality in Chilean Purebred Stallions: Effect of Age and Seasonality. In this work, we investigated the influence of age and seasonality on sperm motility and DNA fragmentation in post-thawing semen from Chilean Purebred Stallions (CPS), a horse breed presenting the oldest genealogy record in South America with an interesting reproductive industry. Despite that semen from aged CPS is frozen all year round, there is a lack of studies characterizing the breed semen freezability in accordance with age and seasonality. Twenty fertile CPS were grouped into the young group, the middle group, and the aged group. Ten ejaculates from each stallion were obtained by using ...
Data set of the proteome of fresh and frozen thawed stallion spermatozoa. This paper provides the dataset of proteins of stallion ejaculates before and after cryopreservation. The data report the analysis and identification of stallion sperm proteins obtained from the same ejaculates and split in two subsamples. The first aliquot consisted on fresh spermatozoa and the second aliquot was frozen and thawed spermatozoa. Samples were analyzed using a UHPLC/MS/MS system consisting of an Agilent 1290 infinity series UHPLC coupled to an Agilent 6550 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). We provide a data set of 2226 different proteins, with ...
Effects of cashew gum and nanoparticles on cooled stallion semen. Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Gums have been suggested as an alternative cryoprotectant to glycerol for stallion spermatozoa. Therefore, the present experiment was designed to verify whether the effect of addition of cashew gum (CG), or nanoparticles (NP) containing CG, to the extender before cooling on sperm quality in stallion semen. Ejaculates from 6 stallions were extended and split between six treatment groups (control, a-tocopherol [TOC], CG1, CG0.5, NP1 and NP0.5), stored in cryotubes ...
Equine seminal plasma and sperm membrane: Functional proteomic assessment. During ejaculation, a large amount of seminal plasma proteins interact with the sperm membrane, leading to a series of biochemical and structural changes implicated in sperm function and gamete interaction. However, the roles of the majority of these proteins remain unknown. This study aimed to investigate the proteome and functionality of the major equine proteins of seminal plasma and the sperm membrane. Seminal plasma and enriched-membrane proteins (150 μg) were separated by two-dimensional gel electrophoresis, and the respective maps were analyzed. Protein identification was performed by...
Efficiency of Semen Cryopreservation in Stallions. Differences in the cryotolerance of spermatozoa exist among stallions, but it remains to be determined to what extent such differences are affected by breed. In this study, post-thaw semen quality in stallions presented for semen cryopreservation was analysed retrospectively (1012 ejaculates from 134 stallions of 5 breeds). The percentage of frozen-thawed ejaculates acceptable for artificial insemination (AI) and the number of insemination doses per cryopreserved ejaculate was calculated. Logistic regression analysis revealed sperm motility in raw semen as the most important explanatory variab...
Comparative Semen Microbiota Composition of a Stallion in a Taylorella equigenitalis Carrier and Non-Carrier State. Contagious equine metritis is receiving renewed attention due to the continuous detection of carriers in apparent agent-free farms. Interactions of Taylorella with the seminal microflora may be the plausible cause behind these spontaneous changes of the carrier state. Accordingly, the aim of this study was to compare the differences in the seminal microbiome composition of one stallion in the contagious equine metritis carrier state and non-carrier state. Samples were cryopreserved after their extraction. Cell disruption was performed by high-speed homogenization in grinding media. Bacterial f...
Stallion Semen Cooling Using Native Phosphocaseinate-based Extender and Sodium Caseinate Cholesterol-loaded Cyclodextrin-based Extender. The objective of this study was to compare semen parameters and embryo recovery rates of cooled stallion semen extended with INRA 96 or BotuSemen Gold. In experiment 1, 45 ejaculates from nine mature stallions were collected, assessed, and equally split between both extenders and then extended to 50 million sperm/mL. Then, the extended semen was stored in three passive cooling containers (Equitainer, Equine Express II, and BotuFlex) for 48 hours. In experiment 2, the same ejaculates extended in experiment 1 were cushion-centrifuged, the supernatant was discarded, and the pellets were resuspend...
Practical experience with artificial insemination (AI) using fresh chilled and frozen semen in mares. 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...
Laboratory Production of Equine Embryos. Assisted reproduction technologies (ART) are well developed in humans and cattle and are gaining momentum also in the equine industry because of the fact that the mare does not respond to superovulation but can donate large numbers of oocytes through ovum pick up (OPU). After collection, the oocytes can be fertilized by intracytoplasmic sperm injection (ICSI) using a variety of stallion semen samples, even of poor quality, and the resulting embryos can establish high pregnancy rates after cryopreservation and transfer. The discoveries that equine oocytes can be held at room temperature without...
The Effect of Different Vitrification and Staining Protocols on the Visibility of the Nuclear Maturation Stage of Equine Oocytes. In this study, we compared two staining protocols assessing the nuclear chromatin stage of equine oocytes after vitrification using permeable and nonpermeable cryoprotectants. Slaughterhouse-derived oocytes (n = 155) were obtained from a total of 32 mares and in vitro matured in M199 medium for 42 hours at 38.5°C in 5% CO2. In the first experiment, two concentrations of Hoechst 33342 (HO) were tested (10 μg/mL; P1 and 2.5 μg/mL; P2) combined with 50 μg/mL of propidium iodide as staining protocols to evaluate the visibility of matured oocytes (n = 44). In the second experiment, 111 o...
Proteomic profiling of stallion spermatozoa suggests changes in sperm metabolism and compromised redox regulation after cryopreservation. Proteomic technologies allow the detection of thousands of proteins at the same time, being a powerful technique to reveal molecular regulatory mechanisms in spermatozoa and also sperm damage linked to low fertility or specific biotechnologies. Modifications induced by the cryopreservation in the stallion sperm proteome were studied using UHPLC/MS/MS. Ejaculates from fertile stallions were collected and split in two subsamples, one was investigated as fresh (control) samples, and the other aliquot frozen and thawed using standard procedures and investigated as frozen thawed subsamples. UHPLC/M...
Effect of relaxin on semen quality variables of cryopreserved stallion semen. The aim of the study was to ascertain effects of different concentrations of relaxin added to extender medium during the pre-freezing incubation periods on quality variables of stallion frozen-thawed spermatozoa. Semen samples collected from three stallions were filtered, diluted with skim milk, and centrifuged at 600g for 10 min. Sperm pellets were suspended in BotuCrio freezing medium to a final concentration of 50 × 10 sperm/mL. The diluted semen was divided into five experimental groups supplemented with 0 (control), 12.5, 25, 50, or 100 ng/mL of relaxin. The semen samples were transferre...
Clinical Application of in Vitro Embryo Production in the Horse. The first reports of in vitro embryo production (IVEP) by conventional in vitro fertilization and intracytoplasmic sperm injection in horses date respectively from approximately 30 and 25 years ago. However, IVEP has only become established in clinical practice during the last decade. The initial slow uptake of IVEP was largely because the likelihood of success was too low to make it an economically viable means of breeding horses. During the last decade, the balance has shifted, primarily because of significant improvements in the efficiency of recovering immature oocytes from live donor m...
Effect of warming method on embryo quality in a simplified equine embryo vitrification system. Equine embryo vitrification is still not a well-established technique in equine practice. Notably, little work has been done on the effect of the warming system on viability of vitrified embryos. Our goal was to evaluate the effect of warming without cryoprotectants on in vitro - produced (IVP) embryo viability in culture, quality assessment parameters, and pregnancy after transfer. Equine IVP blastocysts were vitrified using commercial embryo vitrification media and a semi-closed vitrification device. In Exp. 1, we evaluated two warming temperatures (room temperature, RT, ∼22 °C; and 38...
Cryopreservation of equine oocytes: looking into the crystal ball. Invitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of invivo-matur...