Influence of Seminal Plasma Antioxidants and Osteopontin on Fertility of the Arabian Horse

This study was designed to investigate enzymatic antioxidants’ activity and nonenzymatic antioxidants’ levels in seminal plasma of stallions and to relate them with season, age, and fertility of stallions. Fifty ejaculates were collected from six healthy Arabian stallions, 4-22 years old. Ejaculates were evaluated by conventional methods. Five milliliters of each semen sample was centrifuged, and the supernatant seminal plasma was stored at −20°C. Five antioxidants, in addition to osteopontin (OPN) and testosterone, were determined in stallion seminal plasma by using commercial enzyme-linked immunosorbent assay kits. Results revealed that uric acid, ascorbic acid, OPN, and testosterone concentrations and glutathione peroxidase (GPx) activity in stallions’ seminal plasma were high (P < .05) during spring. GPx activity was higher (P < .05) in age group B (11-18 years old) than in age group A (4-10 years old). The effect of stallions’ age on GPx activity in the fertility groups was highly significant (P < .01). OPN concentration was highest (P < .05) in age group A. Uric acid and OPN concentrations and GPx activity in stallions’ seminal plasma and percent of sperm motility were higher (P < .05) in fertility group III (>70%) than in fertility group I (<50%). However, ascorbic acid concentration, catalase activity and percentage of sperm abnormalities were lower (P < .05) in fertility group III than in fertility group I. It was concluded that season and stallion age may affect antioxidant defense systems in stallions’ seminal plasma. The impairment of seminal antioxidants and OPN could lead to low fertility.     

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.     

Quality of Raw and of Cold-Stored Semen in Icelandic Stallions

The aim of the present study was to evaluate the quality of raw and cooled semen in Icelandic stallions. Experiments were performed using seven stallions aged between 3 and 19 years. From each stallion, six ejaculates were collected, and semen quality was determined. Thereafter, the semen was split into eight equal parts and processed with and without centrifugation using the extenders INRA 82-egg yolk, INRA 96, GENT, and Equi-Pro to a final concentration of 30 × 10⁶ sperm/mL. The extended semen was then cooled in an Equitainer, where it was stored for 24 hours, and subsequently refrigerated for another 24 hours at 5°C. Immediately after dilution as well as after 24 and 48 hours storage, sperm motility was analyzed using computer-assisted sperm analyzer, and viability was assessed after dual DNA staining with SYBR-14 in combination with propidium iodide. The results show that the stallion had a significant (P < .05) influence on all variables evaluated in raw semen, and mean (±SEM) values of 43.4 ± 4.3 mL for the volume, 193.0 ± 17.0 × 10⁶ sperm/mL for the concentration, 6.7 ± 0.5 × 10⁹ for total sperm and 73.5 ± 2.1% for total sperm motility, 48.7 ± 2.0% for progressive motility, and 65.3 ± 2.0% for rapid cells were measured. In the cold-stored semen, all variables were significantly (P < .05) influenced by the stallion, extender, and storage time (48 hours). Except for Equi-Pro, all extenders examined were suitable for cooled semen preservation. For storage of more than 24 hours, centrifugation and removal of the seminal plasma were advantageous for all extenders with the exception of Equi-Pro.     

The Effects of Centrifuged Egg Yolk Used with INRA Plus Soybean Lecithin Extender on Semen Quality to Freeze Miniature Caspian Horse Semen

The aim of this study was to determine the synergistic effects of centrifuged egg yolk (EY) and soybean lecithin on post-thaw Caspian horse sperm motility, morphological abnormalities, and assessment of membrane integrity. The centrifuged EY (CEY) was added at concentrations of 2% and 4% to a defined INRA plus 1.25% soybean lecithin extender used to freeze Caspian horse semen. In this experiment, ejaculates collected from each Caspian horse (n = 4) were divided into three equal aliquots and diluted in CEY 2% (INRA2), 4% (INRA4) supplemented, and without any CEY (INRA0) in INRA plus 1.25% soybean lecithin extender, respectively. Thereafter, samples were frozen and thawed following a standard protocol. Sperm cryosurvival was evaluated in vitro by microscopy assessments of post-thaw sperm motility (by means of computer-assisted semen motility analysis [CASA]), acrosomal and other abnormalities (head, mid-pieces, and tail) and plasma membrane integrity (evaluated by HOST). In Caspian stallion, semen extended with INRA2 had significantly higher CASA motility and CASA progressive motility than those extended with the rest of extenders after freezing and thawing (P < .001). There was no significant difference in path velocity (VAP), VCL, and ALH among three groups (P > .05). For straight line velocity (P < .01) and LIN (P < .001), the highest values were obtained from the INRA4 group. The highest percentages of acrosomal and other abnormalities were found in semen diluted in INRA4 (P < .001). In the group frozen INRA2, the percentage of membrane integrity was significantly higher than that of the other groups (P < .001). The use of CEY 2% in combination with soybean lecithin significantly improved Caspian horse semen freezability.     

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.     

Relationship of Sperm Quality to Fertility after 4 Days of Cooled Storage of Equine Semen

This study evaluated measures of sperm quality in relation to fertility achieved with fresh semen or semen cooled and stored. Semen from 1 stallion was collected and processed to provide 3 treatments: group 1 received fresh semen; group 2 received cooled semen containing 50% seminal plasma (SP) stored for 4 days; and group 3 received cooled semen containing 50% SP stored for 1 day, then centrifuged and resuspended in fresh extender containing 10% SP on days 1 to 3. Inseminates were evaluated for sperm motion characteristics and the percentage of sperm with intact membranes (SMI). Mares (n = 34) in estrus were treated with an ovulation-inducing drug and inseminated with 100 million membrane-intact sperm on the following day. Pregnancy status was determined via transrectal ultrasonography 2 weeks after ovulation. The mean percentage of SMI was higher in group 1 (81%, initial) than in group 2 (74%, day 4) or group 3 (74%, day 4) (P < .05). The median percentages of total sperm motility differed among the groups (77%, 5%, 59% for groups 1, 2, and 3 respectively; P < .05). Median values for the percentages of progressively motile sperm and curvilinear velocity for group 1 (55%, 216 μm/s) and 3 (37%, 186 μm/s) were higher than for group 2 (1%, 73 μm/s) (P < .05). Pregnancy rates did not differ among groups (5 of 11, 45% in group 1; 5 of 11, 45% in group 2; and 7 of 12, 58%, in group 3; P = .77). These data suggest that, at least for this stallion, sperm membrane integrity may be a more valuable means of assessing potential fertility of cooled-stored semen than sperm motion characteristics.     

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, 2, and 3 mg/mL CLC to obtain 120 × 10⁶ sperm/mL spermatozoa concentration. The semen was cryopreserved using customized freezing protocols. Evaluation of seminal parameters, the C:P ratio, and the oxidative status of jack spermatozoa was analyzed at all stages of cryopreservation. The oxidative status in the jack semen was evaluated by measuring malondialdehyde, glutathione and total antioxidant capacity levels. The results indicated that the mean percent values for various seminal quality parameters and the oxidative parameters were found to be significantly higher (P < .05) in CLC-treated groups with the highest values for 2 mg of CLC/120 × 10⁶ spermatozoa. In conclusion, the present study revealed that the supplementation of CLC before cryopreservation has significantly reduced the oxidative stress and also increased the C:P ratio during semen cryopreservation process. Furthermore, a reduction in lipid peroxidation levels, reduced damage to the sperm plasma and acrosome membranes and improvement in the post-thaw sperm integrity as well as stability were recorded.     

Cryopreservation of Cooled Semen and Evaluation of Sperm Holding Media for Potential Use in Equine-Assisted Reproduction Procedures

Processing stallion semen for assisted reproductive procedures, such as intracytoplasmic sperm injection (ICSI), requires special considerations regarding cooling, concentrating, and handling of sperm. The aim of experiment 1 was to determine whether cooled semen could be frozen without removal of seminal plasma and at a low sperm concentration while maintaining motile sperm for ICSI selection procedures. In experiment 2, five media for holding stallion sperm were compared to evaluate sperm motility for an interval of time sufficient for ICSI sperm selection procedures. In experiment 1, semen samples from eight stallions were cooled for 24 hours in two extenders, CST (E-Z Mixin-CST “Cool-Store/Transport” Animal Reproduction Systems) and INRA96 (Institut National de la Recherche Agronomique, IMV International Corporation), before being frozen in four freezing diluents, and were evaluated at 0, 45, and 75 minutes after thawing. The cooling extender did not significantly affect sperm motility, but modified French and glycerol egg yolk diluents provided the best sperm motility for frozen–thawed groups. In experiment 2, semen samples from seven stallions were used to test five media for holding sperm. Samples were analyzed for total and progressive motility at hourly intervals. Mean total and progressive motility were not different (P > .05) among groups from 1 through 4 hours. At 5 hours, groups differed (P = .004), with sperm held in Tyrode’s with albumin, lactate, and pyruvate having higher (P < .05) total and progressive motility than all other samples. In conclusion, motile stallion sperm can be obtained after the sperm are cooled for 24 hours, frozen, and thawed; various media are available to maintain sperm motility during equine ICSI selection procedures.     

Sodium Caseinate and Cholesterol Improve Bad Cooler Stallion Fertility

This study aimed to assess the effects of sodium caseinate and cholesterol to extenders used for stallion semen cooling. Two ejaculates from 19 stallions were extended to 50 million/mL in four different extenders and cooled-stored for 24 hours at 5°C. The extender 1 (E1) consisted of a commercially available skim milk–based extender. The extender 2 (E2) consisted of E1 basic formula with the milk component being replaced by sodium caseinate (20 g/L). The extender 3 (E3) consisted of E1 basic formula added to cholesterol (1.5 mg/120 million sperm). The extender 4 (E4) consisted of a combination of the E2 added to cholesterol. At 24 hours after cooling, sperm motility parameters, plasma membrane stability (PMS), and mitochondrial membrane potential were assessed. In addition, cooled semen (1 billion sperm at 5°C/24 hours) from one “bad cooler” and one “good cooler” stallions, split into four extenders was used to inseminate 30 light breed mares (30 estrous cycles/extender). Milk-based extenders (E1 and E2) had superior sperm kinetics than E3 and E4 (P < .05). Plasma membrane stabilization was significantly higher (P < .05) in E4 than E1, whereas E2 and E3 presented intermediate values (P > .05). The mitochondrial potential intensity was lower (P < .05) in E2 and E4 groups compared with E1 and E3. The good cooler stallion had high fertility (∼80%) in all extenders. However, for bad cooler stallion, E1 40% (8/20) and E2 45% (9/20) had poor fertility (P < .05) compared with E4 85% (17/20), whereas E3 55% (11/20) had intermediate value (P > .05). In conclusion, the association of sodium caseinate and cholesterol improved fertility of bad cooler stallion semen cooled for 24 hours.     

The Effect of Cholesterol Addition,Buffer, and pH on Equine Sperm Stored at 5°C

Cooled stallion semen has a short viable life, which ranges with acceptable motility and viability from 24 up to 48 hours. The purpose of this study was to compare the effects of storage pH, the ability of three different zwitterionic buffers, and cholesterol-loaded cyclodextrins (CLC) to preserve the motility and integrity of stallion sperm cooled to 5°C for 48 hours. Fourteen ejaculates were collected and split to receive CLC or not (control group). After incubation, each sample was split into six subsamples and diluted in KMT extender containing 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), or 2-(N-morpholino)ethanesulfonic acid (MES) buffers, and the final pH was adjusted to either 7.0 or 6.6, totalizing 12 experimental groups as a function of CLC, buffer, and pH variables (2 × 3 × 2 factorial). The motility parameters and integrity of plasma and acrosome membranes (live cell index) were determined using computer-automated semen analysis and epifluorescence microscopy at 3, 6, 24, and 48 hours of cooling period. According to results, pH was not a significant source of variation for motility and live sperm over different cooling periods. However, samples diluted in BES exhibited higher progressive motility within 3 hours and higher percentages of total motile cells after 48 hours of incubation at 5°C (P < .05). After 24 hours of storage, CLC-treated sperm samples presented higher motility than control group (P < .05), and after 48 hours of incubation, CLC-treated sperm exhibited higher percentages of live, motile, and progressively motile sperms (P < .05). We inferred that equine semen diluted in KMT containing BES as buffer and CLC treatment improve the equine sperm survival during storage at 5°C for 48 hours.     

Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze) with seminal plasma had any impact on the quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples coming from separate stallions were used and different functional parameters (sperm membrane integrity and lipid disorder, motility, intracellular Ca²⁺ levels, and intracellular concentrations of peroxides and superoxides) were evaluated after incubation with or without 50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on sperm function and survival. The third experiment was an in vivo study. Twenty-five mares were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were significantly (P < .05) higher than those observed when seminal plasma was not infused (64% vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can be maintained at 4°C for up to 96 hours. In addition, not only does supplementing frozen-thawed epididymal sperm with seminal plasma have any damaging effect on their quality but it may also improve pregnancy rates after artificial insemination.     

The effects of equine somatotropin on pituitary and testicular function in the stallion during the nonbreeding season

An experiment was conducted to determine the effects of equine somatotropin on the reproductive axis of the stallion during the nonbreeding season. Adult stallions were treated with equine somatotropin (20 μg/kg body weight [BW]; n = 5) or saline (n = 4) daily for 21 days starting in January. During the last week of treatment, stallions were subjected to low- and high-dose injections of luteinizing hormone (LH), as well as low- and high-dose injections of gonadotropin-releasing hormone (GnRH) and thyrotropin-releasing hormone (TRH). Two months after the onset of somatotropin treatment, semen was collected from all stallions every other day for 14 days. Treatment with equine somatotropin increased (P < .001) daily IGF-1 concentrations but had no effect (P > .1) on concentrations of LH, follicle-stimulating hormone (FSH), or testosterone. The testosterone responses to injections of LH were similar (P > .1) between treatments. Likewise, the LH, FSH, prolactin, and testosterone responses to the injections of GnRH/TRH were similar (P > .1) between groups. At seminal collections, stallions treated with somatotropin exhibited greater volumes of gel-free semen (P < .01) and gel (P < .05) and had decreased time until ejaculation (P < .05). In conclusion, somatotropin treatment for 21 days may alter the long-term accessory gland contribution to seminal volume but does not appear to alter pituitary gonadotrope function or testicular testosterone secretion.     

The Effect of 2-Hydroxypropyl-β-Cyclodextrin on Post-Thaw Parameters of Cryopreserved Jack and Stallion Semen

Cyclodextrins improve post-thaw viability and motility of semen as well as mediate cholesterol efflux and subsequent acrosome reaction in spermatozoa from several species. The objectives of this study were: (a) to assess the effect of prefreeze addition of 60 mM hydroxypropyl-β-cyclodextrin (β-CD) on post-thaw viability and motility of jack and stallion semen cryopreserved in ethylene glycol-based freezing extenders containing 5% or 20% (v/v) egg yolk (LEY and HEY, respectively), and (b) to evaluate the ability of 1 μM calcium ionophore A23187 and/or 60 mM β-CD to induce acrosome reaction in thawed jack and stallion spermatozoa. Post-thaw motility of spermatozoa cryopreserved in HEY was higher (P < .05) for jack but lower (P < .05) for stallion spermatozoa when compared with LEY. Jack and stallion spermatozoa both exhibited higher (P < .05) motility when cryopreserved in 60 mM β-CD than without β-CD. Curvilinear velocity was faster (P < .05) for jack and stallion spermatozoa cryopreserved in LEY than in HEY. A treatment × time interaction affected (P < .05) the proportion of spermatozoa that underwent acrosome reaction. Post-thaw incubation of jack and stallion spermatozoa with β-CD for 90 minutes induced acrosome reaction in 85% and 22% of viable sperm cells, respectively; however, only 32% of jack and 8% of stallion spermatozoa incubated with calcium ionophore underwent acrosome reaction. This study is the first to evaluate the effect of β-CD (not loaded with cholesterol) on jack semen cryopreservation, and results reveal that β-CD may be a useful tool to enhance semen cryopreservation and to induce post-thaw acrosome reaction in jack spermatozoa.     

The Effect of Trehalose Supplementation of INRA-82 Extender on Quality and Fertility of Cooled and Frozen-Thawed Stallion Spermatozoa

Stallion semen cryopreservation is often associated with poor post-thaw sperm quality. Sugars act as nonpermeating cryoprotectants. The aim of the present study was to evaluate the cryoprotective effect of trehalose on stallion sperm quality and field fertility rates subjected to cooling and freeze–thaw process. Semen samples were collected from six Arabian stallions, divided into five different treatments in a final concentration of 100 × 10⁶ sperm/mL by using INRA-82 extender containing 0, 25, 50, 100, and 200 mM of trehalose then subjected to both cold storage and cryopreservation. Sperm motility, acrosome, plasmatic membrane, and DNA integrity were analyzed, and 57 mares were used to evaluate the field fertility of chilled and frozen-thawed semen. Results showed that the extender containing 100 mM trehalose only increased the functional acrosomal, plasma membrane, and DNA integrities. The inclusion of 50 mM trehalose in semen extender resulted in significantly (P < .05) increased post-thaw total motility compared to the control group, and chilled semen achieved higher pregnancy rates compared to the frozen-thawed one. Pregnancy rate of mares inseminated with frozen-thawed semen (P < .05; 46.15% vs. 36.36%, respectively) was lower than those inseminated with chilled semen (76.47% vs. 68.75%, respectively) but higher than control. In conclusion, addition of 50 mM trehalose yielded the highest quality stallion semen after cooling and post-thawing in terms of motility, integrities of acrosome, membrane, and DNA as well as improved field fertility.     

Quality of Fresh,Cooled, and Frozen Semen From Stallions Supplemented with Antioxidants and Fatty Acids

This study assessed the effect of oral supplementation with the primary antioxidants and fatty acids involved in spermatogenesis (L-carnitine, selenium, vitamin E, omega-3, and omega-6) on the seminal quality in fresh, cooled, and frozen semen of stallions (n = 8), using a randomized design. The animals were divided into Group I (n = 4) and Group II (n = 4) for a 30-week experiment. The two groups alternated between nutraceutical supplementation and a placebo over the course of the experiment. Semen collections were performed in two sets: once in the middle of the experiment, before the two groups switched treatments, and once at the end. The volume, appearance, sperm concentration, spermatozoa kinetics, and membrane integrity of fresh semen were evaluated. The spermatozoa kinetics and membrane integrity of cooled (for 24, 36, and 48 hours) and frozen semen were also evaluated. No differences were observed in volume, appearance, and sperm concentration between treatment and control. However, compared with placebo, nutraceutical supplementation increased (P < .05) total motility, trajectory speed, as well as plasma and acrosomal membrane integrity in spermatozoa from fresh semen. In cooled semen, nutraceutical treatment also increased (P < .05) total motility, speed, and membrane integrity of spermatozoa compared with the control. In frozen semen, supplementation increased (P < .05) spermatozoa progressive motility and plasma membrane integrity. Our results suggest a positive, synergistic effect of the antioxidant L-carnitine and selenium on spermatozoa kinetics. Similarly, the increase in plasma and acrosomal membrane integrity could be attributed to higher concentrations of polyunsaturated fatty acids (a key cell-membrane component), combined with the prevention of excess lipid peroxidation by antioxidants. In conclusion, supplementation with nutraceuticals containing fatty acids and antioxidants improved the quality of fresh, cooled, and frozen stallion semen. Therefore, nutraceutical use should increase the success of artificial insemination with cooled and cryopreserved semen.     

Combined cryopreservation of canine ejaculates collected at a one-hour interval increases semen doses for artificial insemination without negative effects on post-thaw sperm characteristics

A limiting factor in canine artificial insemination (AI) is the low number of insemination doses obtained per ejaculate. In this study, semen was collected from dogs (n = 28) either once and frozen directly after collection or the same dogs were submitted to a dual semen collection with a 1-hr interval and the two ejaculates were combined for cryopreservation. We hypothesized that combining two ejaculates increases semen doses per cryopreservation process without negative effects on semen characteristics. Total sperm count was lower in semen from a single semen collection in comparison with the combination of the first and second ejaculate of a dual semen collection (p < .001). The percentage of motile and membrane-intact spermatozoa determined by computer-assisted sperm analysis (CASA) in raw semen did not differ between single and combined dual ejaculates and was reduced (p < .001) by cryopreservation to the same extent in single (motility 73.7 ± 1.8%, membrane integrity 65.6 ± 2.2%) and combined dual ejaculates (motility 72.7 ± 2.3%, membrane integrity 64.6 ± 2.5%). The percentage of spermatozoa with morphological defects increased after cryopreservation (p < .001) but was similar in single and combined dual ejaculates. The CASA sperm velocity parameters decreased with cryopreservation (p < .001) but did not differ between single and combined dual ejaculates. The number of insemination doses increased from 2.7 ± 0.4 for single to 4.7 ± 0.8 for combined dual ejaculates (p < .01), based on 100 million motile spermatozoa per frozen-thawed semen dose. In conclusion, combining two ejaculates collected at short interval for one cryopreservation process increases the number of AI doses without compromising semen quality.     

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 resuspended at 100 million sperm/mL with their respective extender. Semen was then cooled and stored as in experiment 1. In both experiments, sperm motility parameters, plasma membrane integrity, and high mitochondrial membrane potential were assessed at 0, 24, and 48 hours post cooling. For experiment 3, 12 mares (n = 24 cycles) were bred with 48 hour–cooled semen from one stallion. Semen was processed as described in experiment 1. Mares had embryo flushing performed by 8-day post-ovulation. In experiment 1, BotuSemen Gold displayed superior total and progressive motility relative to INRA 96 (P < .05). There were no significant differences between the types of containers in any experiment. In experiment 2, INRA 96 and BotuSemen Gold extenders had similar total and progressive motility, but BotuSemen Gold had superior sperm velocity parameters at all timepoints. Embryo recovery was identical for both extenders (50%). Finally, the results obtained herein suggest that BotuSemen Gold is a suitable alternative to be included in semen cooling tests against INRA 96 in clinical practice.     

Application of Techniques for Sperm Selection in Fresh and Frozen-Thawed Stallion Semen

The objective of this research was to improve the techniques in processing chilled and frozen‐thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim‐up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb^®) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 12, 773) were selected for processing complete ejaculates either for cooled‐storage or cryopreservation. In a third experiment (Exp. III), pregnancy rates from inseminations with semen processed according to the techniques tested in Exp. II were compared with those obtained with semen processed according to routine procedures. In Exp. I (six stallions, six ejaculates/stallion), between 48 and 92% of spermatozoa were lost following the different sperm selection procedures (p < 0.05). Preparation of sperm increased percentage of progressively motile spermatozoa (pms) [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] and decreased percentage of sperm head abnormalities [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] probably by not improving the quality of individual cells, but by elimination of spermatozoa of inferior quality. In Exp. II (eight stallions, three ejaculates/stallion) Leucosorb^® and GWS procedures allowed the filtration of large volumes (extended ejaculates) for routine laboratory practice. GWS and Leucosorb^® filtration resulted in increased motility, membrane integrity and sperm viability after storage of spermatozoa until 48 h at +5°C when compared with control (diluted) and centrifuged semen (p < 0.05). Significantly more spermatozoa were recovered after centrifugation (87.8 ± 15.4%) compared with GWS (63.5 ± 18.6%) and Leucosorb^® filtration (53.6 ± 22.3%). GWS or Leucosorb^® procedure resulted in successful cryopreservation of stallion semen without centrifugation for removal of seminal plasma. The per cycle conception rate of inseminated mares using 200 × 10⁶ pms transferred within 8 h after collection of semen was not affected by GWS filtration or Leucosorb^® separation when compared with centrifugation (n.s.; Exp. III). In conclusion, GWS and Leucosorb^® filtration results in the improvement of semen quality and should be considered as a method for stallion semen processing. Additional studies are needed for the evaluation of potentially higher fertilizing ability of stallion spermatozoa separated by techniques for sperm selection.     

Alkaline and Acid Phosphatase Activity, pH and Osmotic Pressure of Boar Semen

Alkaline phosphatase activity was recorded in forty ejaculates of the sperm rich fraction of boar semen as 9,790 ± 5,250 Klein-Babson-Read units per 100 ml. of seminal plasma. Acid phosphatase activity in the same ejaculates was 681 ± 304 Babson-Read units per 100 ml. of seminal plasma. No alkaline phosphatase activity was detected in the seminal plasma of vasectomized boars.

The pH of the sperm rich fractions was 7.69 ± 0.33 and the osmotic pressure was 313.56 ± 7.98 milliosmols.

     

The Relationship of Mitochondrial Membrane Potential,Reactive Oxygen Species,Adenosine Triphosphate Content,Sperm Plasma Membrane Integrity,and Kinematic Properties in Warmblood Stallions

Equine sperm possesses a unique physiology because its energy supply is mostly dependent on oxidative phosphorylation of mitochondria as an aerobic source of adenosine triphosphate (ATP) generation. The present study was, therefore, conducted to investigate the relationship between sperm kinematic and functional variables in stallions. Semen samples were collected from five warmblood stallions (three ejaculates from each stallion), diluted with INRA96 and transferred to the laboratory. Next, sperm motility, mitochondrial membrane potential (MMP), production of superoxide anion (as a reactive oxygen species; ROS), ATP content, and plasma membrane integrity were assessed. Motion and functional characteristics differed among investigated stallions (P < .05). In addition, it was revealed MMP was positively correlated with the level of ROS and ATP content and progressive motility (P < .05). The level of ROS was positively correlated with ATP content and negatively correlated with plasma membrane integrity and straightness (P < .05). Adenosine triphosphate content was positively correlated with progressive motility, curvilinear velocity, average path velocity, and beat cross frequency and reversely correlated with plasma membrane integrity and straightness (P < .05). Plasma membrane integrity was positively correlated with straight line velocity, linearity, and straightness and negatively correlated with curvilinear velocity (P < .01). In conclusion, the present study substantiated that kinematic and functional characteristics varied among various warmblood stallions. Furthermore, the present study implicated although higher mitochondrial activity increases ATP synthesis, it leads to elevated superoxide anion production, which could culminate in disintegration of the sperm plasma membrane, thereby altering motion characteristics and swimming pattern of sperm.