Cryopreservation Of Stallion Spermatozoa Research Articles

Improving storage stability of cryopreserved stallion sperm for storage at elevated subzero temperatures

Improving storage stability of cryopreserved stallion sperm for storage at elevated subzero temperatures

Cryopreservation of equine spermatozoa reduces plasma membrane integrity and phospholipase C zeta 1 content as associated with oocyte activation.

Phospholipase C zeta (PLCZ1) is considered the major sperm-borne oocyte activation factor. Cryopreserved stallion spermatozoa are commonly used for intracytoplasmic sperm injection (ICSI). However, plasma membrane damage and protein modifications caused by cryopreservation could impair sperm structure and function, leading to a reduction of PLCZ1 and oocyte activation after ICSI. We compared membrane integrity and PLCZ1 abundance in populations for fresh, frozen, and refrozen stallion spermatozoa, either thawed and refrozen at room or low temperature; and examined the effect of relative PLCZ1 content on cleavage after ICSI. Western blotting, ELISA, and immunofluorescence were conducted in stallion spermatozoa, freezing extenders, and detergent-extracted sperm fractions to detect and quantify PLCZ1. Retrospectively, PLCZ1 content and cleavage rate were analyzed. Fresh, frozen, and refrozen at room and low temperatures spermatozoa were evaluated for acrosomal and plasma membrane integrity and PLCZ1 content using flow cytometry. Western blotting, ELISA, and immunofluorescence revealed significant reduction of PLCZ1 in spermatozoa after cryopreservation and confirmed PLCZ1 detection in extenders. After detergent extraction, a PLCZ1-nonextractable fraction remained in the postacrosomal region of spermatozoa. Plasma membrane integrity was significantly reduced after freezing. Acrosomal and plasma membrane integrity were similar between frozen and refrozen samples at low temperature, but both were significantly higher than samples refrozen at room temperature. Acrosomal and plasma membrane integrity significantly correlated to PLCZ1 content. Percentages of PLCZ1-labeled spermatozoa and PLCZ1 content were reduced after freezing but not after refreezing. Relative content and localization of PLCZ1 were associated with cleavage rates after ICSI. Sperm PLCZ1 content associates with cleavage rates after ICSI. Cryopreservation is detrimental to sperm plasma membrane integrity and PLCZ1 retention. However, refreezing did not result in additional PLCZ1 loss. Refreezing stallion spermatozoa at a low temperature resulted in better survival but did not improve PLCZ1 retention.

The effect of controlled stress on kinematic parameters of cryopreserved stallion sperm

Semen cryopreservation is an important tool for genetic improvement of horse breeds and the reproductive efficiency of stallions. The freezing and thawing process can cause injuries and damage to sperm structures which may impair fertility. The use of controlled stress has been shown to be an alternative for cellular protection against cryoinjuries (Priebenszky et al., Biology of Reproduction. 2010;83:690-697). Thus, this study aimed to evaluate whether the submission of sperm to controlled stress using different negative pressures can improve sperm kinetics. Four healthy stallions aged between 6 and 20 years old were used. Semen was collected (n = 12 ejaculates) and extended with a skim-milk based extender (Botusemen, Botupharma) 1:1 and then transported to the laboratory in a cooling device (Botuflex, Botupharma). The negative pressure exposure technique followed the protocol described by Casali et al., (Acta Scientiae Veterinariae. 2014; 42: 1240) adapted using a vacuum pump (Vix® ¼ hp 60Hz) coupled to a pressure chamber. Each ejaculate was divided and exposed to 4 treatments (Control - no pressure; P200 - submission to a negative pressure of 200 mBar/5min; P500 - negative pressure of 500 mBar/5min; P800 - negative pressure of 800 mBar/5min), centrifuged at 600 gfor 10 min and the pellet was cryopreserved with Botucrio (Botuphatma) at 100 million/ml. For the evaluation of sperm kinetics, computer-assisted sperm analysis (CASA; Androvision software; Minitube) was used. Data were analyzed by ANOVA (SPSS statistical package) followed by Tukeytest (p<0.05). Total motility (Control: 45.9±14.1%; P200: 43.18±10.8%; P500: 45±12.1%; and P800: 41.2±13.2%), circular motility (Control: 1.9±1.2%; P200: 2.3±0.9%; P500: 2.4±1.0%; P800: 1.6± 1.2%), rapid motility (Control: 6.7± 3.7%; P200: 5.5±1.8%; P500: 6.0±4.2%; P800: 4.1±2.3%), slow motility (Control: 20.3±10.2%; P200: 17.4±6.4%; P500: 16.8±7.4%; P800: 15.3±8.9%),non-progressive motility (Control: 16.8±6.5%; P200: 17.9±5.9%; P500: 18.2 ±6.6%; P800: 19.9±8.1%) and progressive motility (Control: 28.7±13.6%; P200: 23.7±9.2%; P500: 26.3±9.5%; P800: 20.7±10.7%) were similar between negative pressure treated and control groups. These findings suggest that negative pressure does not affect sperm motility. However, the effect of negative pressures with the aim to protect sperm against cryo-injures requires further exploration. In conclusion, submission of equine sperm to a controlled stress using different negative pressures does not impair sperm kinetics.

Effect of Testosterone, Dihydrotestosterone, Estradiol and Cortisol on the Quality of Fresh and Cryopreserved Stallion Sperm

The effect of steroid hormones on the quality of fresh and cryopreserve sperm has not been fully understood. This study aimed to evaluate the effect of testosterone, dihydrotestosterone, estradiol, and cortisol on the quality of fresh and cryopreserved stallion sperm. The study was conducted on 40 Equus caballus stallions, including Arab (n=20), Oryol trotting (n=4), Standardbred (n=4), and Soviet Heavy Draft (n=12) breeds. The average age of the experimental animals was 9.9 ± 0.7 years. We determined standard quality indicators in fresh and cryopreserved sperm and the concentration of steroid hormones in the blood plasma of stallions. Results of the study suggested a negative correlation between the level of testosterone with total (r=-0.41; p&lt;0.01) and progressive (r=-0.44; p&lt;0.01) sperm motility in cryopreserved sperm as well as in fresh sperm (r=-0.38; p&lt;0.05 and r=-0.39; p&lt;0.05 correspondingly). While the level of estradiol showed a positive correlation with survival rate in cryopreserved (r=0.35; p&lt;0.05) and in fresh (r=0.33; p&lt;0.05) sperm. Further, the level of cortisol in the blood plasma of stallions did not show any statistically significant correlations with the qualitative characteristics of sperm. A positive relationship was found between the concentration of dihydrotestosterone with the volume of ejaculate (r=0.37; p&lt;0.05) and the total number of sperm in the ejaculate (r=0.43; p&lt;0.01). Results of the study can be concluded that steroid hormones have different effects on the quality indicators of fresh and cryopreserved sperm of stallions and their concentration in the blood should be considered when selecting stallions for cryopreservation of sperm.

The inhibition of spermatic cystine/glutamate antiporter xCT (SLC7A11) influences the ability of cryopreserved stallion sperm to bind to heterologous zonae pellucidae

Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfasalazine (SS), a specific inhibitor of SLC7A11 antiporter. Viability, motility, immunolocalization of tyrosine phosphorylated proteins and the ability to bind to heterologous zonae pellucidae were evaluated. Thawed sperm from seven stallions (2 ejaculates/stallion) was washed and resuspended in Tyrodes media; each thawed ejaculate was divided in Control (CTR) and 3 samples supplemented with: 0.5 mM Cystine (CysS), 500 μM Sulfasalazine (SS) and 0.5 mM CysS + 500 μM SS (CysS + SS). After 1 h of incubation at 37 °C, samples were washed twice, resuspended in capacitating BWW medium and incubated at 38 °C under 5% CO2. After 30 and 60 min, sperm motility, viability and tyrosine phosphorylated protein immunolocalization, used as capacitation status index, were evaluated. After 30 min of capacitation, 4 × 105 sperm were co-incubated with denuded pig oocytes in capacitation medium for 30 min for the heterologous binding assay. None of the sperm parameters studied (motility, viability and tyrosine phosphorylation) showed any difference respective to control. The number of sperm bound per oocyte (mean ± SEM) tended to increase in CysS group (44.0 ± 12.3) respect CTR (40.8 ± 10.8) while decreased in SS group (32.4 ± 7.8) (p < 0.01). Moreover, CysS + SS group showed a lower binding rate (32.0 ± 10.0) compared to CysS (p < 0.001). Our results suggest that CysS supplementation of thawed stallion sperm can influence their ability to bind to heterologous zona pellucidae as the inhibition of CysS incorporation by SLC7A11 reduced the number of sperm bound per oocyte. This effect does not seem to be ascribed to a modification of sperm motility, membrane integrity and tyrosine phosphorylation.

Coenzyme Q-10 improves preservation of mitochondrial functionality and actin structure of cryopreserved stallion sperm.

Coenzyme Q-10 (CoQ-10) is a cofactor for mitochondrial electron transport chain and may be an alternative to improve sperm quality of cryopreserved equine semen. This work aimed to improve stallion semen quality after freezing by adding CoQ-10 to the cryopreservation protocol. Seven saddle stallions were utilized. Each animal was submitted to five semen collections and freezing procedures. For cryopreservation, each ejaculate was divided in three treatments: 1) Botucrio® diluent (control); 2) 50 μmol CoQ-10 added to Botucrio® diluent; 3) 1 mmol CoQ-10 added to Botucrio® diluent. Semen batches were analyzed for sperm motility characteristics (CASA), plasma and acrosomal membranes integrity and mitochondrial membrane potential (by fluorescence probes propidium iodide, Hoechst 33342, FITC-PSA and JC-1, respectively), alterations in cytoskeletal actin (phalloidin-FITC) and mitochondrial function (diaminobenzidine; DAB). The 1 mmol CoQ-10 treatment presented higher (P<0.05) amount (66.8%) of sperm cells with fully stained midpiece (indicating high mitochondrial activity) and higher (P<0.05) amount (81.6%) of cells without actin reorganization to the post-acrosomal region compared to control group (60.8% and 76.0%, respectively). It was concluded that the addition of 1 mmol CoQ-10 to the freezing diluent was more effective in preserving mitochondria functionality and cytoskeleton of sperm cells submitted to cryopreservation process.

PSV-39 The relationship between structural integrity and sperm survival during hypothermic storage of stallion sperm

Abstract The aim of this work was to study the relationship between ultrastructural integrity and sperm survival during hypothermic storage of chilled and cryopreserved stallion sperm. Semen from 37 stallions, were collected during the breeding season. The structural integrity of sperm was studied using a Hitachi 700 electron microscope. Progressive motility was evaluated using an Olympus BX 41 phase contrast microscope. Sperm survival was defined as the ability of spermatozoa to maintain progressive motility above 5% during hypothermic (+4C) storage of sperm. Spearman nonparametric correlation analysis was used. Sperm survival at a temperature +4°C in chilled semen averaged 115.4±11.47 hours. After freezing sperm survival decreased on average to 48.9±5.56 hours (P &amp;lt; 0.001). A reliable correlation between the number of sperm with intact heads and sperm survival at a temperature of + 4°C in diluted (r=0.60; P &amp;lt; 0.001) and thawed (r=0.63; P &amp;lt; 0.001) sperm was established. A significant negative correlation between the number of spermatozoa with degradation of the acrosome in native sperm and their survival (r=-0.48; P &amp;lt; 0.01) under the hypothermic storage was found. For cryopreserved semen, a similar dependence has not been established. A significant correlation between the number of spermatozoa with the normal position of the acrosome and the sperm survival rate in native sperm was also established (r=0.52; P &amp;lt; 0.01). A significant negative correlation between sperm with acrosome hypoplasia and sperm survival was found (r=-0.67; P &amp;lt; 0.001). We assume that the structural integrity and ability of spermatozoa to maintain progressive motility for a long time under conditions of hypothermic storage depends on the quality of spermatogenesis and the resistance of cells to low and ultra-low temperatures. Authors acknowledge financial support from Russian Science Foundation, Grant No: 20-16-00101, development program of Bioresource collections “Cryobank of genetic recourses the All-Russian Research Institute for Horse Breeding.”

Peculiarities of collection and cryopreservation of stallion sperm

Horse breeding in Ukraine has always been an industry of national importance, the functional orientation of which has changed because of the development of socioeconomic relations. Currently, the role and importance of horses in state farms is complex nature. Breeding horses are used to improve existing and create new, more advanced breeds that would meet the requirements of European and world standards. Improving the efficiency of reproduction technologies in horse breeding and further progress of breeds is impossible without the implementation of methods of artificial insemination of horses, including cryopreservation of sperm. Multiple improvements have been achieved in the past 10 years with regard to utilization and processing of frozen stallion semen, use of alternative cryoprotectors and new commercially available extenders that allow beƩer cell protection during freezing. It is analyzed that there is only one licensed cryogenic sperm bank of certified sports stallions in Ukraine, which is located in Zhashkiv Equestrian School, from where sperm is transported abroad. It contributes to the improvement of the existing gene pool of horses in our country and promotes the industry. The technological process of selection and cryopreservation of sperm in breeding stallions of the Belgian sport breed in the Zhashkiv Equestrian School of Cherkasy region is investigated. The latest methods for selection and cryopreservation of stallion sperm are presented. It has been investigated that cryopreservation using pre-cooling of sperm for 2.5 h and subsequent rapid freezing in liquid nitrogen vapors is an effective method for maintaining the fertilizing ability of stallions. It is established that following the above rules of selection, evaluation, calculation, dilution, packaging, cryopreservation, and storage of sperm of high-value stallions, the number of sperm banks might be increased. This popularizes the industry, allows to increase the number of farms for breeding horses, and to develop scientific developments in biotechnology and reproduction of these animals.&nbsp

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 parameters were analyzed immediately after light-stimulation (0 min) and after 120 min of incubation at 38 °C. Sperm motility was evaluated using a Computerized Semen Analysis System (CASA), and flow cytometry and different fluorochromes were used to evaluate the integrity and lipid disorder of plasma membrane, mitochondrial membrane potential and intracellular levels of peroxides and superoxides. Irradiation significantly increased the percentages of spermatozoa with high mitochondrial membrane potential (1-1-1 pattern) and the intracellular levels of peroxides (2-2-2 pattern) at 0 min. In addition, sperm kinematic parameters (2-2-2 and 3-3-3 patterns) and percentages of viable spermatozoa with low membrane lipid disorder (3-3-3 pattern) were significantly higher in irradiated samples than in the control at 120 min. Our results indicate that red-light stimulation could help increase the resilience of frozen-thawed stallion sperm to withstand post-thaw incubation at 38 °C for 120 min and that these effects rely on the irradiation pattern. Further research should evaluate whether light-stimulation could also have a positive on fertility rates after artificial insemination.

Cryopreservation of stallion spermatozoa with different glycerol concentrations

Cryopreservation of stallion spermatozoa with different glycerol concentrations

Centrifugation Force and Time Alter CASA Parameters and Oxidative Status of Cryopreserved Stallion Sperm.

Conventional sperm selection techniques used in ARTs rely on centrifugation steps. To date, the different studies reported on the effects of centrifugation on stallion sperm motility provided contrasting results and do not include effects on mitochondrial functionality and different oxidative parameters. The effects of different centrifugation protocols (300× g for 5′, 300× g for 10′, 1500× g for 5′ and 1500× g for 10′ vs. no centrifugation) on motility and oxidative status in cryopreserved stallion sperm, were analyzed. After centrifugation, almost all motility parameters were significantly altered, as observed by computer-assisted sperm analysis. A polarographic assay of oxygen consumption showed a progressive decrease in mitochondria respiration from the gentlest to the strongest protocol. By laser scanning confocal microscopy, significant reduction of mitochondrial membrane potential, at any tested protocol, and time-dependent effects, at the same centrifugal force, were found. Increased DNA fragmentation index at any tested protocol and time-dependent effects at the same centrifugal force were found, whereas increased protein carbonylation was observed only at the strongest centrifugal force. These results provide more comprehensive understandings on centrifugation-induced effects on cryopreserved stallion sperm and suggest that, even at a weak force for a short time, centrifugation impairs different aspects of equine sperm metabolism and functionality.

Evaluation of Fertilization Capacity of Cryopreserved Stallion Sperm, Directly After Thawing and After Cooled Storage

SexedULTRA™ is an improved method of sex-sorting sperm creating a less damaging environment to retain sperm integrity through the sorting process. The aim of this study was to evaluate the in vitro characteristics of fresh and frozen bovine sperm using the SexedULTRA™ method, and compare it to conventional (non-sorted) sperm. For both methods, percent total sperm motility was estimated visually and also classified into total and progressively motile using a computer assisted sperm analyzer (CASA). Percent sperm with intact plasma membranes (VIA) and acrosomes (PIA) were assessed using flow cytometry and sperm DNA fragmentation index (DFI) was estimated using the Bull sperm Halomax® Kit. Two contemporaneous ejaculates from 10 bulls were processed and cryopreserved using one of the two procedures (SexedULTRA™ and conventional). Sperm motility, VIA and PIA were assessed post-thaw (0 h) and post-incubation (3 h at 37 °C, 8 h and 24 h at 18 °C). DFI was analyzed post-thaw (0 h) and after 6, 24, 48 and 72 h of incubation at 37 °C. In a second experiment, ejaculates from 7 bulls were split sampled into the two types of processing (SexedULTRA™ and conventional) and diluted using a fresh semen extender developed for sex-sorted bovine sperm. Sperm quality was assessed after dilution (0 h) and after incubation for 12, 24, 48, 72 h at 18°, and the same time points of incubation at 37 °C for DFI. Frozen-thawed SexedULTRA™ sperm was significantly (P < 0.05) better than conventional semen after a 3 h incubation at 37 °C for PIA, and after a 24 h incubation at 18 °C for percent visual motility and PIA. DFI was significantly lower for SexedULTRA™ compared to conventional at all time points of incubation (37 °C). Fresh SexedULTRA™ sperm showed improved quality compared to conventional at all time points of incubation at 18 °C for percent visual and total motile sperm, VIA, PIA, and DFI. Significant differences were also found in progressive motile sperm immediately after dilution (0 h), but not at any time point after incubation. The results show that the SexedULTRA™ process maintains the quality of sex-sorted sperm and, in many cases, has better in vitro longevity than conventional semen.

Seminal plasma influences the fertilizing potential of cryopreserved stallion sperm

Seminal plasma (SP) contains proteins that may influence cryosurvival and prevent capacitation-like changes due to freezing and thawing. The objective of this study was to investigate the effect of adding pooled SP from “good” (GF) or “bad” (BF) freezer stallions on sperm cells' fertilizing ability. “Good freezers” refers to stallions that usually produce ejaculates which can withstand cryopreservation, whilst “bad freezer” stallions produce ejaculates which cannot tolerate the freezing process. A heterologous zona binding assay with in vitro matured bovine oocytes was used to assess the binding ability of equine sperm cells as a possible alternative to artificial insemination trials. The effect of adding SP i) prior to cryopreservation; ii) after thawing of sperm cells selected by single layer centrifugation (SLC); iii) to capacitation medium, was evaluated. Adding SP from GF stallions prior to cryopreservation reduced the mean number of sperm cells bound to the zona pellucida (ZP) compared to control (P = 0.0003), SP-free sperm cells and group received SP from BF stallions (P ≤ 0.0001 for both). After thawing SLC-selected sperm cells treated with 5% SP showed a decrease in binding ability compared with SP-free sperm cells (P ≤ 0.0001). The binding affinity of sperm cells was higher in the group treated with SP from GF than with SP from BF stallions (P ≤ 0.05). Prolonged exposure to SP impaired the ability of stallion sperm cells to undergo capacitation and bind to ZP, regardless of the source of SP (P ≤ 0.0001). The response of equine sperm cells to SP is influenced by the ability of the sperm cells to withstand cryopreservation and is affected by the timing of exposure and the origin of SP. Customization of the protocol for individual stallions is recommended to optimize the effect.

An endeavor to improve longevity of cryopreserved equine sperm

Exposure of sperm cells to the oxidative stress pending hypothermic storage of semen has been suggested to be responsible, in part, for the decline of their motility and fertility. This study was conducted to evaluate the in-vitro effects of antioxidants (AOs) and / or caffeine on longevity of cryopreserved stallion spermatozoa. Aliquots from the gel-free fraction of semen ejaculates (n=12), collected from 5 Arabian stallions (9-18 years old) of unknown sperm freezability, were mixed 1:1 with a Tris-egg yolk extender (TEYE), centrifuged at 500 χ g for 5 min and sperm cells were frozen in the form of 0.25-ml concentrated pellets after 2-step addition of TEYE supplemented with or without AOs (0.50 mg /ml Na pyruvate, 1 mg / ml Na thiosulfate, 5 mg / ml bovine serum albumin, 0.15 mg / ml zinc chloride and 0.50 mg / ml ferulic acid). The final pre-freeze concentrations of glycerol and sperm cells were 5% and 562-924 χ IO6 / ml, respectively. Frozen pellets from non-AOs and AOs-treated sperm were thawed in a Tris-citric acidglucose solution (40°C) containing 0, 0.49, 0.97 or 1.94 mg / ml caffeine and incubated (140-230 χ IO6 sperm / ml) at 30°C for 3 h. Sperm progressive motility (%) was assessed after centrifugation, before freezing and after 0, 1, 2 and 3 h of thawing. The results revealed significant (P&lt;0.05) effects of sperm treatments only on post-thaw motility. Neither AOs alone nor caffeine alone could significantly ameliorate the maintenance of sperm motility. AOs plus 0.97 or 1.94 mg / ml caffeine were the superior supplements in improving the longevity of stallion spermatozoa.

Effects of Two Amino Acids on Motion Parameters and Enzymatic Antioxidant Activity of Freeze-Thawed Stallion Spermatozoa

In this study, effects of amino acids, glutamine and proline, as supplements of freezing extender were studied during cryopreservation of stallion spermatozoa. The CASA and biochemical assays were performed following thawing of sperm that had been frozen in basal medium as control (E1) and basal medium containing 3-mM (E2), 30-mM (E3), and 60-mM proline (E4); 5-mM (E5) and 50-mM glutamine (E6); and 3-mM proline plus 5-mM glutamine (E7). E2 increased progressive motility (P < .001), motile spermatozoa (P > .05), values of curvilinear velocity, average path velocity, mean angular displacement (MAD), lateral head displacement, beat cross-frequency, and straightness (STR) compared to control extender. E3 led to a less motile and progressive motile sperm. E3, E4, and E6 were found to provide for lower mean linearity (LIN), wobble, and STR values than control group. E5 increased LIN value and decreased MAD value significantly (P < .05). CASA parameters of E7 were not different with control. The levels of malondialdehyde did not change. The glutathione peroxidase activity remained unchanged with E2, E3, E4, and E5 but decreased in the E6 (1,933 ± 210.1) and E7 (1,976.5 ± 5.8), although the change was not significant (P > .05). E3 (2,080 ± 288) and E5 (1,553.3 ± 155.8) significantly increased catalase activity (P < .05), and superoxide dismutase percent inhibition with E7 (38.46 ± 28.2) was significantly higher than other groups (P < .05). The higher motility of stallion spermatozoa in diluent containing 3-mM proline may allow for the more efficient use of frozen-thawed stallion semen for insemination and could contribute to the improvement of semen cryopreservation in the world horse industry.

Quercetin reduces oxidative stress after cryopreservation of stallion spermatozoa

Quercetin reduces oxidative stress after cryopreservation of stallion spermatozoa

Field fertility of liquid stored and cryopreserved flow cytometrically sex-sorted stallion sperm.

The fertility of sex-sorted, cryopreserved stallion sperm must be improved for the sex-sorting technology to be applied commercially. To optimise the conditions used to liquid store stallion sperm prior to sex-sorting and assess the fertility of sperm following sex-sorting and cryopreservation. Both in vitro experiment and randomised controlled trial in healthy, client-owned mares. Stallion ejaculates (n = 9) were diluted in either a skimmed milk (KMT) or BSA (I-BSA) based media to 25 × 106 sperm/ml directly (+SP25) or washed to remove seminal plasma and diluted to 25 or 111 × 106 sperm/ml (-SP25 and -SP111). Sperm were stored for 18 h at 10 to 15°C and -SP25 and +SP25 treatments were centrifuged and resuspended to 111 × 106 sperm/ml. Sperm were incubated under H33342 staining conditions and motility, viability and acrosome integrity assessed. Semen was collected from stallions (n = 4), liquid stored at 10-15°C for up to 5 h and sperm either cryopreserved directly, sex-sorted and cryopreserved, or sex-sorted and returned to liquid storage until insemination. Low-dose hysteroscopic insemination was performed in 23 mares randomly allocated to the semen preparation group and pregnancy determined following embryo flushing on Day 9 after ovulation, or via transrectal ultrasonography on Day 14 after ovulation. Skimmed milk was superior to I-BSA in maintaining motility, viability and acrosome integrity. Seminal plasma removal did not affect the parameters measured at the concentrations examined. Conception rates did not differ significantly between the groups, although a high incidence of pregnancy loss was observed in both the cryopreserved groups. While the conception rates achieved are among the highest yet reported for sex-sorted, cryopreserved stallion sperm, the high incidence of pregnancy loss suggests that the development of the resulting embryos was significantly impaired by the sperm processing treatments.

Improved techniques for freezing stallion semen as assessed by post-thaw parameters

INRA96 has been used successfully as a base extender for equine frozen semen [1-4]. The development of INRA96 [5] was a major step forward as an effective extender that used native phosphocaseinate. Independently, INRA96 (INRA96) was glycerolized [2, 3] and then augmented with the addition of egg-yolk [6, 7] to freeze stallion sperm cells. The objective of this research was to investigate the cryopreservation of stallion sperm using various percentages of glycerol and egg yolk. For the study, a wide range of stallion breeds (8) and ages (2 to 21 years) were used.

Quercetin improves the postthaw characteristics of cryopreserved sex-sorted and nonsorted stallion sperm

Excessive reactive oxygen species generation during sex sorting and cryopreservation of stallion sperm leads to DNA fragmentation, lipid peroxidation, and motility loss. In this study we investigated whether antioxidant supplementation during sex sorting and cryopreservation could ameliorate the effects of reactive oxygen species on stallion sperm. In experiment 1, the postthaw characteristics of stallion sperm (N = 9) cryopreserved in the presence or absence of catalase (200 U/mL), cysteine (0.2 mg/mL), or quercetin (0.15 mM) was examined. Motility and acrosome integrity were assessed at 0, 1, and 3 hours after thawing. The sperm chromatin structure assay (SCSA; detectable DNA fragmentation index [DFI], mean DFI, and DFI) was used to assess DNA integrity immediately after thawing. Quercetin increased the total postthaw motility (25.3% vs. 20.9%; P < 0.05), but there was no beneficial effect of catalase or cysteine. Based on these results, the effect of quercetin during cryopreservation on the postthaw zona binding ability of sperm was assessed using a heterologous (bovine) zona binding assay. Quercetin increased the number of sperm bound per oocyte (13.6 vs. 9.2; P < 0.05) compared with the control. In experiment 2, the effect of quercetin (0.15 mM) in the media used during semen storage and transport, Hoechst 33342 staining and cryopreservation of stallion sperm (N = 9) was investigated. Motility, acrosome integrity, and viability were assessed at 0, 1, and 3 hours after thawing and SCSA was performed at 0 hours after thawing. Quercetin supplementation during sex sorting and cryopreservation improved DNA integrity (SCSA; detectable DFI of 54.9% vs. 74.6%, P < 0.05; mean DFI of 270.2 vs. 288.1, P < 0.05; and DFI of 26.3% vs. 28.5%, P < 0.05) compared with control sex-sorted sperm. There was no beneficial effect of quercetin on the motility, acrosome integrity, or viability of sex-sorted sperm. In conclusion, quercetin significantly improved the motility and zona binding ability of cryopreserved stallion sperm, and reduced DNA fragmentation in sex-sorted, cryopreserved stallion sperm.

Delayed cryopreservation of stallion sperm: effect of iodixanol density gradient centrifugation

Delayed cryopreservation of stallion sperm: effect of iodixanol density gradient centrifugation