Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid

The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 10⁶/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 10⁶ ± 21, 59 × 10⁶ ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.     

Effect of Centrifugation Technique on Post-storage Characteristics of Stallion Spermatozoa

Three ejaculates were collected from four stallions and used to compare the effects of three centrifugation methods on post-storage motility and recovery of available sperm. Two aliquots per ejaculate were diluted with skim milk-glucose (SKMG) extender to 50×10⁶ sperm/mL, placed in 50-mL conical bottom tubes, and centrifuged at either 700g for 15 minutes (700g) or 600g for 12 minutes (600g). A third aliquot was diluted 1:1 with SKMG, placed in 15-mL conical tubes, and centrifuged at 400g for 7 minutes (400g). Subsamples from each pre-treated diluted ejaculate were held at room temperature and evaluated for motility at the same time as the post-centrifugation pre-storage motility evaluation was made for treated aliquots. After centrifugation, samples from each aliquot were stored at 5°C for evaluation after 24 and 48 hours or frozen in liquid nitrogen. Percentage of available sperm harvested was higher (P ≤ .05) for aliquots centrifuged in 15-mL tubes at 400g versus 600g in 50-mL tubes. After centrifugation, total but not progressive motility of aliquots centrifuged at 700g was lower than that for noncentrifuged controls and sperm from aliquots centrifuged at 400g in 15-mL tubes. After cold storage, values for total but not progressive motility or velocity were higher (P ≤ .05) for aliquots centrifuged in 15-mL tubes at 400g compared with those centrifuged in 50-mL tubes at both 600g and 700g. Postthaw motility of frozen sperm was not different between centrifugation treatments. Poststorage percentages of intact acrosomes and detached heads did not differ because of centrifugation treatment.     

Single-Layer Centrifugation Through Colloid Positively Modifies the Sperm Subpopulation Structure of Frozen–Thawed Stallion Spermatozoa

The present study attempted to select the subpopulation of stallion spermatozoa that best survived a conventional freezing and thawing procedure, using centrifugation of post-thawed semen samples through a single layer of a glycidoxypropyltrimethoxysilane-coated silica colloid with a species-specific formulation (Androcoll-E™). After freezing and thawing, four sperm subpopulations were identified, listed as FT1 to FT4. While subpopulations FT1 and FT2 were characterized by low sperm velocity, high velocities characterized the ones called FT3 and FT4. The single-layer centrifugation (SLC)-handled sperm sample was enriched in subpopulation FT3, reaching a proportion of 82.6% of the present spermatozoa, in contrast with the non-filtered control post-thawed semen, where this sperm subpopulation only accounted for 16.3% of the total. It is concluded that in the equine industry, the SLC is a practical, easy-to-perform approach to improve the quality of equine frozen–thawed semen samples.     

Stallion Sperm Integrity After Centrifugation to Reduce Seminal Plasma Concentration and Cool Storage for 4 days

The objective of the study was to investigate if reducing the seminal plasma of stallion extended semen by centrifugation once will suffice to maintain acceptable semen quality for insemination after 4 days of cool storage. Collected semen was extended to 25 × 10⁶ sperm/mL and subjected to one of the following treatments: noncentrifuged (control), centrifuged for 10 minutes at 900 × g and 1800 × g. The supernatant was partially removed, and the sperm pellet, reconstituted and re-extended. It was then placed in a passive cooling device overnight and then transferred to a refrigerator for the remainder of the cooling period. At day 0, 2, and 4, total motility (TM), progressive motility (PM), and plasma (PLM) and acrosomal membrane integrity were assessed. Centrifuged groups had higher TM and PM at day 4 than the control group (P < .05). Likewise, centrifuged groups had higher intact PLM in day 4 (P < .05). A single centrifugation cycle to reduce seminal plasma concentration will suffice to preserve sperm integrity acceptable for an artificial insemination dose up to 4 days of cool storage.     

Colloidal Centrifugation of Stallion Semen Results in a Reduced Rate of Sperm DNA Fragmentation

Stallion spermatozoa recovered and examined immediately after colloidal centrifugation resulted in a higher straight‐line velocity (VSL) than sperm processed using direct conventional centrifugation (p = 0.000), but there was no differences in the progressive motility or sperm DNA fragmentation (SDF) as determined by the sperm chromatin dispersion assay. However, when centrifuged spermatozoa were incubated at 37°C for 24 h to determine the rate of SDF (r‐SDF), a lower r‐SDF (p = 0.0011) was observed in those sperm recovered after colloidal separation (0.5 ± 0.1%/h) compared to direct (1.2 ± 0.4%/h) or no centrifugation (r‐SDF = 1.2 ± 0.3%/h). These results confirm that colloidal separation of stallion spermatozoa results in prolonged sperm DNA longevity, but these differences were only apparent following a period of incubation and dynamic assessment. Consequently, we strongly recommend the use of the dynamic form of the SDF assay for evaluating centrifugation and/or other ex vivo procedures, as a single basal assessment of SDF may inadvertently result in a false‐positive evaluation of DNA quality.     

Effect of Removing Seminal Plasma Using a Sperm Filter on the Viability of Refrigerated Stallion Semen

Cooling of equine semen obtained from some stallions results in lower seminal quality and viability when the seminal plasma (SP) is present. The objective of this study was to evaluate the effect of the removal of SP using a Sperm Filter on the viability of cooled stallion semen. For this purpose, 31 stallions were used. Their ejaculates were divided into three groups: CN, semen was diluted with an extender; FLT, SP was removed by filtration; and CT, SP was removed by centrifugation and cooled to 15°C for 24 hours. Sperm kinetics and plasma membrane integrity were evaluated immediately after collection (T0) and after 24 hours of refrigeration (T1). No difference (P > .05) was noted at T1 for total sperm motility (TM), progressive sperm motility, or plasma membrane integrity when semen samples from all the stallions were analyzed. However, when samples from stallions termed “bad coolers” were analyzed (TM = <30% at T1), a difference was observed in TM and progressive sperm motility for CN compared with FLT and CT at T1. Sperm recovery was greater when SP was removed using the filter (FLT) to that when the SP was removed by centrifugation (CN) (89% vs. 81%). Thus, we concluded that filtering with a Sperm Filter is an efficient and practical method for removal of SP from stallion ejaculates, with lower sperm loss than centrifugation. We also found that the presence of SP reduces the quality and viability of cooled semen from stallions whose semen is sensitive to the process of refrigeration.     

Effects of the Addition of Autologous Seminal Plasma to Highly Concentrated Stallion Semen After 48 Hours of Cooled Storage

Insemination with chilled transported semen has become distinctly important in the horse-breeding industry. To ensure cell survival during cooled storage, semen is diluted with an appropriate extender and the concentration of seminal plasma (SP) is reduced. Nevertheless, SP plays an important immunomodulatory role in the female genital tract and supports sperm fertility. The aim of the present study was to evaluate the effect of the addition of autologous SP after cooled storage to highly concentrated stallion semen. Therefore, SP was removed by simple centrifugation of extended semen, aspiration of the supernatant, and resuspension of the sperm pellet with semen extender. Motion characteristics were evaluated after cooled storage for 48 hours at concentrations of 333 × 10⁶ sperm/mL in comparison with stored samples at concentration of 25 × 10⁶ sperm/mL (control). The highly concentrated semen samples were diluted with an extender containing 0%, 5%, 20%, and 80% SP directly before motility analysis. Dilution of the cooled semen with a fresh semen extender without SP (0%) increased kinematic parameters (curvilinear velocity [VCL] 137.3 vs. 151.8; straight-line velocity [VSL] 49.0 vs. 57.5; average path velocity [VAP] 69.5 vs. 79.4 μm/second; amplitude of lateral head [ALH] 3.1 vs. 3.3 μm; beat cross frequency [BCF] 31.6 vs. 33.5 Hz; P < .05) but not total motility (51% vs. 43%) and progressive motility (46% vs. 36%) compared with controls. The addition of SP after storage for 48 hours decreased sperm total motility and progressive motility regardless of SP concentration: 5 (38% and 34%), 20 (37% and 33%), and 80% SP (27% and 22%; P < .05). In contrast, kinematic parameters were enhanced by extenders containing 5% and 20% SP (VCL: 148.0 and 155.6; VSL: 59.2 and 60.9; VAP: 78.7 and 81.9; BCF: 33.4 and 35.7; ALH: 3.4 and 3.4; P < .05). However, using an extender containing 80% SP was detrimental to kinematic parameters (VCL: 151.2; VSL: 52.2; VAP: 76.9; BCF: 34.8; P < .05) except for ALH, which increased (3.5; P < .05). In conclusion, cooled storage at concentrations of 333 × 10⁶ sperm/mL did not affect sperm motility. The addition of a fresh extender or an extender containing small concentrations of SP to highly concentrated ejaculated sperm increased kinematic values after storage; however, increasing concentrations of SP decreased sperm motility.     

A Comparison of the Ability of Three Commercially Available Diluents to Maintain the Motility of Cold Stored Stallion Semen

The objective of this study was to compare the ability of three commercially available extenders to promote poststorage motility of stallion spermatozoa stored at 5°C with and without centrifugation to remove the seminal plasma. Diluents tested included skim milk glucose (SKMG), INRA 96, and VMD-Z. All diluents were tested with (-SP) and without (+SP) centrifugation to remove most of the seminal plasma. In experiment I, after 48 and 72 hours of storage, total (TM) and progressive (PM) motility values were higher (P ≤.05) for those aliquots subjected to the INRA 96-SP as compared with either SKMG treatment. After 72 hours of storage, PM of spermatozoa stored in VMD-Z-SP was superior to that of spermatozoa stored in SKMG regardless of the presence of seminal plasma (P ≤.05). In the second experiment, after 48 hours of storage, PM of spermatozoa subjected to the INRA 96-SP and VMD-Z-SP treatments were superior (P ≤.05) to those for all treatments that had been stored without removal of seminal plasma. Removal of the seminal plasma and resuspension of the sperm pellet with either INRA 96 or VMD-Z resulted in TM after 48 hours of storage that were similar to those obtained after 24 hours of storage.     

Dietary Polyunsaturated Fatty Acid Supplementation Improves the Quality of Stallion Cryopreserved Semen

The objective was to assess the influence of polyunsaturated fatty acid supplementation on the quality of fresh, cooled, and frozen-thawed stallion semen. Ten stallions received their normal diet (control group) or normal diet plus 150 mL of polyunsaturated fatty acid (PUFA) linseed-based oil (PUFA group). Semen was collected every 15 days during 60 days. Stallions were reversed across the treatments after a sixty-day interval. Semen was evaluated at 2, 6, 12, and 24 hours after cooling and 24 hours after freezing. Motility (MOT), vigor, membrane viability, morphology, acrosome integrity, and osmotic tolerance test (OTT) were evaluated. In the frozen-thawed semen, sperm dynamic characteristics were analyzed by computer-assisted sperm analysis and thiobarbituric acid reactive substances (TBARs) determined. The effects of treatment, time, semen type, and their interactions were submitted to PROCMIX (SAS) and means compared by the Tukey test. There was no treatment effect on the quality of fresh and cooled semen. However, frozen-thawed semen MOT, vigor, and OTT were superior (P < .05) in control compared to PUFA group. An interactive effect of sample day by treatment was observed, such that, TBARs increased over time (P = .002) in the PUFA group after 15, 30, 45, and 60 days from the beginning of supplementation. Thus, sperm may become more susceptible to the reactive oxygen species, probably due to the incorporation of polyunsaturated fat in the cell membrane. The addition of PUFA-enriched oil may be an alternative for improving frozen-thawed semen quality by increasing its MOT and resistance to osmotic changes to which sperm cells are submitted during the freezing process.     

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 stallions, each time. We showed that NA at 20 and 40 mM concentrations could significantly improve the stallion sperm quality markers during cold storage. However, the protective effects were not different between 20 mM and 40 mM concentrations in most measures. Nicotinic acid could also improve the post-thaw stallion sperm quality at 10, 20, and 40 mM concentrations. However, the 40 mM concentration showed a negative impact on some post-thaw kinematic sperm parameters. Nicotinic acid at 10 and 20 mM concentrations could preserve the sperm cryo-tolerance to be frozen up to 8 hours after collection without a significant decline in most of the post-thaw sperm quality measures. Nicotinic acid could also decrease the level of the lipid peroxidation and total reactive oxygen/nitrogen species in the cooled and frozen-thawed spermatozoa, in a dose-dependent manner. Therefore, NA at 20 mM concentration could preserve most of the stallion sperm quality measures during cold storage (42 hours, 5°C) and enabled storage of cooled stallion semen for 6 hours before freezing without significant deterioration of the post-thaw sperm quality.     

Effect of Seminal Plasma Components on the Quality of Fresh and Cryopreserved Stallion Semen

The importance of seminal plasma (SP) components for stallion semen quality and freezability is little known. This study aimed to evaluate the relationship between SP components and fresh/cryopreserved stallion semen quality. Semen of 30 stallions was collected, and then, SP was recovered and lyophilized. Total protein (TP), vitamin C (CVIT), vitamin E (EVIT), vitamin A (AVIT), iron (Fe), copper (Cu), magnesium, and zinc (Zn) in SP were assessed. Sperm was frozen in an extender supplemented with lyophilized SP. In fresh semen motility, abnormal morphology (AM), sperm vitality (SV), and plasma membrane integrity (PMI) were evaluated. In post-thaw semen, additionally, total motility (TM), progressive motility (PM), straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat cross-frequency (BCF) were assessed. Levels of component of SP were established by a distribution analysis. Generalized linear models were fitted. Comparisons of means were done with Tukey's test. Correlation and regression analyses were performed. Vitamins and ions were found to be related to fresh semen quality. For post-thaw sperm, medium TP showed higher semen quality. Negative regression and correlation coefficients between CVIT and all post-thaw semen parameters were found. Low EVIT yielded the lowest PM, VSL, and VAP values, while a high level of AVIT yielded the best results for sperm quality. A high level of Cu yielded higher results for TM, PM, VCL, and ALH. Moreover, a negative correlation was found between Zn, SV, and PMI. In conclusion, SP composition influences fresh and post-thaw stallion semen quality.     

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 ground, and imipramine hydrochloride treatment were performed unsuccessfully. However, a protocol consisted by the association of imipramine (3 mg/kg/v.o.), detomidine (0.02 mg/kg/i.v.), and oxytocin (20 U.I./i.v.) successfully produced ejaculation in this stallion. The semen obtained from ex copula ejaculation of the stallion was collected using a collector cup lined with a plastic bag, which was positioned over the prepuce of the stallion. Semen with good sperm quality (87% of total motility) was obtained using the proposed protocol. Semen was then processed for cryopreservation and post-thawed semen samples presented satisfactory sperm parameters. In conclusion, the association of imipramine, detomidine, and oxytocin can be considered for ex copula semen collection in stallions.     

High or Low Body Fat Deposition in the Presence of a Normal Oral Sugar Test is Not Associated With Postthaw Semen Parameters in Stallions

This study compared the postthaw semen parameters of stallions with high and low body condition score (BCS) and evaluated associations between body morphometric parameters and postthaw semen parameters. Twenty stallions were split into Low BCS (BCS<7, n = 11) and High BCS (BCS ≥7, n = 9) groups, and underwent a complete morphometric analysis (e.g., neck scores and circumference, crest neck height, body weight, and height), and subcutaneous body fat thickness (SFT) at the tail head, withers, shoulders, and retroperitoneal space. A fasted oral sugar test (OST) was conducted on all stallions. One ejaculate from each stallion was frozen with a commercial egg yolk-based extender. Postthaw sperm motility parameters, plasma membrane integrity, mitochondrial membrane potential, hydrogen peroxide and intracellular superoxide production, and lipid peroxidation were analyzed for all stallions. The circumference at 25% and 50% of the neck’s length were larger for High-BCS stallions (P < .05). There were no differences between groups for the neck crest height (P > .05). Stallions with High BCS had greater SFT at the tail head than stallions with Low BCS (P < .05); however, there were no differences between groups in the SFT at the shoulders and withers (P > .05). All stallions had resting blood glucose below the cutoff for equine metabolic syndrome. There were no differences between groups for resting glucose concentrations or for a peak at 30 or 60 minutes after initiation of the OST (P > .05). There were no differences in sperm parameters between groups (P > .05). Collectively, the findings of the present study suggest that High BCS or Low BCS in the presence of normal OST do not explain post-thaw semen parameters.     

Colloidal Centrifugation of Stallion Semen: Changes in Sperm Motility,Velocity, and Chromatin Integrity during Storage

The current study investigated the changes in sperm quality (motility, velocity, and chromatin integrity) occurring during storage at room temperature or 5°C for up to 48 hours in spermatozoa after extension or single-layer centrifugation (SLC) through Androcoll-E. In unselected samples, all parameters of sperm quality deteriorated significantly during storage (P < .01), although the deterioration was faster at room temperature (22–30°C) than for cool storage (P < .01). The SLC-selected spermatozoa had higher motility, velocity, and chromatin integrity than the overall unselected population (motility: selected 85 ± 10%, unselected 56 ± 13%; P < .001; velocity: selected 85.1 ± 13 μm/second, unselected 63.5 ± 15 μm/second; P < .001; and DFI selected 12.2 ± 4.8 μm/second, unselected 23.6 ± 7.4 μm/second; P < .001). Furthermore, sperm quality did not deteriorate with storage in the SLC-selected samples, either at room temperature (22–30°C for 24 hours) or cooled to 4°C (for at least 48 hours), whereas a significant deterioration in sperm quality was observed in the unselected sperm samples (P < .01). Thus, room temperature storage of SLC-selected spermatozoa may be an option for insemination doses from stallions whose spermatozoa do not tolerate cooling. In addition, a new sperm analyzer, the Qualisperm, showed good correlation with subjective motility assessment (r = 0.8, P < .001), was user-friendly, and provided a reasonable volume of data. This instrument may be a useful adjunct to sperm quality assessment at the stud.     

Effect of Different Extenders and Seminal Plasma on the Susceptibility of Equine Spermatozoa to Lipid Peroxidation After Single-Layer Centrifugation, Through Androcoll-E

This study was conducted in an attempt to see whether single-layer centrifugation (SLC) increases the susceptibility of stallion spermatozoa to lipid peroxidation (LPO), in different extenders after removing all seminal plasma (SP). The susceptibility of stallion spermatozoa to LPO was studied before and after SLC. Each ejaculate was split, and aliquots extended with one of the three different extenders: INRA 96, Kenney's, or Equipro, and stored for 24 hours at 5°C (i). From the extended samples, an aliquot was kept as a control and the other was subjected to SLC through Androcoll-E. The selected spermatozoa were re-suspended in the appropriate extenders, without (ii) or with (iii) addition of 50% (v/v) pooled homologous SP for 24 hours at 5°C. Using ferrous sulfate as pro-oxidant, the susceptibility for LPO was flow-cytometrically assessed using the probe Bodipy^581/591-C₁₁. Sperm motility, monitored with a Qualisperm motility analyzer, increased after SLC treatment (P < .001). No significant correlations were found between motility and induced LPO with ferrous sulfate. The SP and extenders, per se, did not have a significant protective effect against LPO, but the interaction between SP and Kenney increased the susceptibility to LPO. However, the selected spermatozoa through Androcoll-E and the subsequent dilution in INRA had a significant protective effect against LPO (P < .05), especially when the oxidative insults were higher (80 μM).     

Motility and Fertility Evaluation of Thawed Frozen Stallion Semen After 24 Hours of Cooled Storage

Breeding mares with cryopreserved semen requires specialized equipment for storage and thawing and more intensive mare management. The objectives of this study were (1) evaluate the longevity of frozen stallion semen once it had been thawed, extended, and maintained at 5°C for 48 hours in a passive cooling container, and (2) determine fertility potential of frozen semen that had been thawed, extended, and used to inseminate mares after 24 hours of cooled storage. Eight ejaculates were collected and aliquots were cooled in either INRA96 and CryoMax LE minus cryoprotectant at a concentration of 50 million total sperm/mL. The remainder of the ejaculate was frozen in CryoMax LE extender at a concentration of 200 million total sperm/mL. Semen was thawed using 1 of 3 thawing protocols, and diluted to a concentration of 50 million total sperm/mL in either INRA96 or CryoMax LE minus cryoprotectant and cooled to 5°C. Sperm motility was evaluated at 24 and 48 hours. Eight mares were inseminated over two estrous cycles using frozen semen that had been thawed, extended in INRA96, and cooled for 24 hours. There was no difference in progressive motility at 24 or 48 hours of cooled-storage post-thaw between the 3 thawing protocols. An overall per cycle pregnancy rate of 56% (9/16 cycles) was achieved using frozen-thawed semen that had been extended and cooled for 24 hours. In summary, frozen stallion sperm was thawed, extended, and cooled to 5°C for 24 hours and still maintained adequate (>30%) sperm motility and fertility.     

Responses of Horses of Various Breeds to a Sympathetic Training Method

The objective of this study was to compare the responses of horses of different breeds to a sympathetic training method used during an initial training period, before the horses started their first racing season. Subject animals were 72 horses: 24 Thoroughbreds, 24 Purebred Arabians, and 24 Angloarabs. Each breed group consisted of 12 colts and 12 fillies. The sympathetic method used to train the horses lasted for 3 days in a row, one session per day. Training was divided into five stages, each stage made up of particular tasks to be achieved. The horse’s reaction to the training was measured with the length of time (seconds) necessary to complete a stage and the horse’s heart rate (bpm) during the stages. Data were analyzed by using multifactorial analysis of variance, taking into account the effect of horse breed, sex, and training session. Results showed that horses of various breeds had different responses to the training. The Angloarabs took the longest to complete the training stages. The 3-day training period caused the highest reaction in the Angloarabs, and the 3-day period was too short to allow for more than half of them to be mounted. Heart rate was more increased in Purebred Arabian and Angloarab fillies than in colts, which shows that fillies require particularly gentle treatment.