Stallion Semen Incubated with Hydrogen Peroxide Decreased DNA Fragmentation as Measured by the TUNEL Assay

Spermatozoa are highly specialized cells that are sensitive to environmental factors such as temperature and redox state. Hydrogen peroxide (H₂O₂) is a reactive oxygen species, and its presence is often associated with a decline in sperm function. The aim of this study was to evaluate the effect of H₂O₂ and heat stress on DNA damage, membrane integrity, and motility of stallion spermatozoa. Spermatozoa from three light-horse stallions were subjected to thermal stress, treatment with H₂O₂, or a combination of both. Treatments were organized using a 2 × 2 factorial design consisting of heat stress (control, 41°C) and potential oxidative stress (control, 50 μM H₂O₂) for 1 hour. The experiments were repeated independently on all stallions. Primary motility parameters were measured using a computer-assisted sperm analysis, whereas DNA damage was assessed using the TUNEL assay. To evaluate membrane integrity, an amine reactive dye was utilized. DNA and membrane integrity were simultaneously assessed in the same flow cytometry assay. Sperm incubated at 41°C were observed to have decreased motility (76.2% vs. 66.6%; P < .05) but not progressive motility (39.2% vs. 25.6%), membrane damaged (30.8% vs. 37.4%), or DNA damage (19.7% vs. 17.3%). Interestingly, when compared to control, treatments with H₂O₂ had decreased DNA damage (19.7% vs. 7.1%; P < .05), but did not affect any other parameter. Although our experiment demonstrated a favorable effect of 50 μM H₂O₂ on DNA damage, further studies need to be conducted to confirm these findings and to clarify any possible signaling mechanisms.     

Effects of Two Different Cooling Devices for Testicles Transport on Stallion Epididymal Sperm Quality

This study evaluates the effects of two cooling devices and temperature for testicles storage on epididymal sperm quality after 24 hours; different levels of seminal plasma (0% and 10%) were evaluated on sperm after recovering. Testicles from six stallions were recovered immediately after castration (2) or at the slaughterhouse (4); of the same animal, one testicle was placed in Equitainer (+8°C), the other in a styrofoam box with ice (+3°C). After 24 hours, the temperature of parenchyma was measured, and testicles and epididymal were weighted. Sperm were flushed from the cauda epididymides with Kenney extender, total sperm number recorded and motility and viability evaluated immediately after flushing (T0) with or without 10% SP (G1 Eq 0%, G2 Eq 10%, G3 Ice 0%, G4 Ice 10%). Motility and viability were evaluated after 24 hours and 48 hours of storage at +4°C. Temperature of the parenchyma was lower in testicles stored in ice compared to Equitainer (3.2 ± 0.6°C and 8.6 ± 2.5°C, respectively; P < .05). Motility and viability at T0 were similar (P > .05) in G1 and G3, whereas addition of SP after recovery significantly improved motility only in samples stored in Equitainer (G2). Viability was higher (P < .05) in G2 than in G4. At T24 and T48, no differences (P > .05) in sperm quality were found between storage methods or samples with or without SP. In conclusion, equine testicles can be safely stored either at lower (+3°C) or higher (+8°C) temperature than +5°C. This can be useful, especially when testicles are shipped in a hot climate, where devices cannot guarantee optimal refrigeration conditions.     

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 Coenzyme Q10 and α-Tocopherol in Skim Milk–Based Extender for Preservation of Caspian Stallion Semen in Cool Condition

The purpose of this study was to determine the effects of different concentrations of coenzyme Q₁₀ (CoQ₁₀) and α-tocopherol (T) along with their interaction effects on the quality of preserved stallion semen at 5°C for a period of 48 hours. Semen was collected and diluted with skim milk–based extender that was supplemented with different antioxidants: no antioxidant (negative control [NC]), 0.9% (vol/vol) dimethyl sulfoxide (positive control [PC]), α-tocopherol (5 [T5] or 10 [T10] mM), CoQ₁₀ (1 [C1] or 2 [C2] μM), 1 μM CoQ₁₀ + 5 mM α-tocopherol (C1T5), 1 μM CoQ₁₀ + 10 mM α-tocopherol (C1T10), 2 μM CoQ₁₀ + 5 mM α-tocopherol (C2T5), and 2 μM CoQ₁₀ + 10 mM α-tocopherol (C2T10), then kept at 5°C. The results showed that C1 extender resulted in higher total motility (62.44 ± 3.82) and plasma membrane integrity (65.16 ± 3.63%) compared with NC after 48 hours of storage (P < .05). Different concentrations of α-tocopherol had no significant effects on sperm quality, with the exception of plasma membrane integrity, compared with NC and PC extenders (P > .05). Also, C1T5 extender improved total and progressive motility, plasma membrane integrity and functionality, and decreased lipid peroxidation compared with NC and C2T10 extenders over 48 hours of storage at 5°C (P < .05). The C1T5 extender was similar to C1 and T5 extenders in all semen parameters evaluated during storage time. In conclusion, between previously mentioned extenders, C1T5 could improve stallion sperm quality during 48 hours of storage. In the present study, none of extenders had effect on sperm quality until 24-hour storage.     

Does Coenzyme Q10 Exert Antioxidant Effect on Frozen Equine Sperm?

During semen cryopreservation, the sensitivity of equine sperm to oxidative stress is increased by the eliminated seminal plasma. Thus, antioxidant addition to the semen extender can be helpful to the sperm survival after freezing and thawing. This work aimed to test whether coenzyme Q10 (CoQ10) added in different concentrations to the INRA 82 freezing extender has antioxidant function on equine sperm to improve its fertilizing ability. Semen samples from five stallions were frozen with the extenders: (T1) INRA 82, control, (T2) T1+ 5 μM CoQ10, (T3) T1+ 25 μM CoQ10, and (T4) T1+ 50 μM CoQ10. After sample thawing, sperm motility and kinetics characteristics were evaluated using a computer-assisted sperm analysis and sperm membrane functionality and integrity were evaluated with a hypo-osmotic swelling test and an epifluorescence microscopy, respectively. The nitrite (NO₂^-) and hydrogen peroxide (H₂O₂) concentrations of the semen samples were measured with spectrophotometry. There was no difference on the sperm characteristics among all treatments (P > .05). However, the 25 μM CoQ10 (T3) decreased NO₂⁻ concentration (6.7 ± 2.2 μM/μg protein) compared with the treatments T1, T2, and T4 (64.3 ± 3.7, 59.4 ± 5.3, 45.1 ± 8.6 μM/μg protein), respectively, as well H₂O₂ concentration (1.8 ± 0.3 μM/μg protein) compared with the control (4.6 ± 0.4 μM/μg protein) and 5 μM CoQ10 treatments (4.8 ± 0.2 μM/μg protein, P < .05). In conclusion, 25 μM CoQ10 plays a significant role as antioxidant to the frozen equine sperm, decreasing NO₂⁻ and H₂O₂ concentrations. Thus, its addition to the INRA 82 freezing extender may be beneficial to the fertilizing ability of equine semen.     

The Use of Cefquinome in Equine Semen Extender

Antibiotics are commonly used in equine semen extender for conservation, if semen has to be stored cooled for a maximum of 48 hours or frozen, to eliminate pathogenic or potentially pathogenic bacteria from semen and reduce the risk of postmating endometritis. Little is known about the effect of antibiotics on spermatozoa when semen is stored over a longer period. Cefquinome, a broad spectrum antibiotic and fourth-generation cephalosporin, has been proven to be a powerful drug for the treatment of endometritis and mastitis in different species. Recently in equine studies, it was found to localize in high concentrations in the endometrium. Therefore, cefquinome was used as the antibiotic in semen extender and compared with a commercial semen extender containing gentamicin for effects on motility and membrane integrity of spermatozoa. During the breeding season, ejaculates from nine light horse stallions were collected and half of each ejaculate was stored for 48 hours in modified Kenney type semen extender containing either cefquinome or gentamicin. At 0, 24, and 48 hours, aliquots (20 μL) of the stored semen were evaluated for (progressive) motility and membrane integrity, as well as for various motility parameters by computer assisted sperm analysis. No differences (P > .05) were found in total motility or progressive motility between extenders at any time point. However, there were differences (P < .05) in velocity parameters, although the effect of velocity parameters on fertility is not clear. In general, semen parameters after storage in non-fat dried skim milk semen extender containing cefquinome are comparable with those after storage in semen extender containing gentamicin. The wider spectrum of bactericidal activity possessed by cefquinome may prove to be beneficial in some cases.     

Effects of Pentoxifylline on Equine Epididymal Sperm

This study evaluated whether pentoxifylline (PTX) present in the flushing extender influenced the function of equine epididymal spermatozoa after recovery and after thawing. For this experiment, 58 testicles from 29 Brazilian Jumping Horses were used. Cauda epididymides of each stallion were separated and flushed with a skim milk extender, with or without 7.18 mM PTX and then subjected to the freezing process. Samples flushed with the extender containing PTX showed a significant increase in total motility, progressive motility, straight line velocity, curvilinear velocity, and percentage of rapid sperm immediately after the recovery of epididymal sperm and after 15 minutes of incubation at 37°C (P < .05). However, the presence of PTX in the flushing extender did not affect the post-thaw motility parameters or plasma membrane integrity (P > .05). The results of this study showed that the PTX present in the flushing extender improved motility parameters of recently recovered epididymal sperm and had no deleterious effects on plasma membrane integrity and freezability of equine epididymal sperm.     

Reproductive efficiency of a new modified boar semen extender for liquid storage

In the present study the Authors developed a new modified boar semen extender for short-term liquid storage, based on the use of amikacin sulphate and fructose rather than gentamicin and glucose. The new extender (ME-S) was evaluated and compared in vitro to commercial ones (CRONOS^™, TRIXcell^™) and to a modified extender designated for long term storage (ME-L) for progressive motility. Progressive motility was not different (P>0.05) among extenders until 120 h of storage, as differences among extenders became significant (P<0.05) at 144 and 166 h. Motility data across time were better for ME-S than TRIXcell^™ (P<0.05). No differences were observed about the morphology and membrane integrity (ORT) among the new extender (ME-S) and the commercial ones. Following the results of the in vitro comparison, an artificial insemination field trial was performed for reproductive efficacy. In this trial ME-L was not used because it was not completely reliable yet. A total of 1011 sows were bred: 506 with ME-S and 505 with a commercial one (CRONOS^™). The pregnancy rate for ME-S was 93.68% (474 pregnant sows), as the commercial extender resulted in 452 pregnancies (89.5%). The statistical comparison was significant (P<0.05) and the number of live piglets born showed an increase of 52.     

Comparison of Various Extenders for Storage of Cooled Stallion Spermatozoa for 72 Hours

Multiple extenders have been developed to preserve cooled stallion semen. Comparisons of some extenders have been made but there is need for further research in this area. Extenders tested included EZ Mixin (Animal Reproduction Systems, Chino, CA), Kenney's, Universal (NASCO, Fort Atkinson, WI), EquiPro, EquiPro CellGuard (Minitube of America, Verona, WI), and INRA 96 (IMV, Maple Grove, MN). Semen was collected and each ejaculate was divided and extended in each of the aforementioned extenders and stored at 4°C. Motility measures were determined using computer-assisted sperm analysis at 0, 24, 48, and 72 hours after collection. Samples were evaluated for total motility, progressive motility (PM), straight-line velocity, curvilinear velocity, straight-line distance, and curvilinear distance. Total motility and PM decreased over time in storage (P < .05). Sperm stored in INRA 96, EquiPro, and EquiPro Cell Guard retained the most total motility and PM over the 72 hour period (P < .05). Universal, EquiPro, and EquiPro Cell Guard had the highest measurements for curvilinear velocity, straight-line velocity, and curvilinear distance (P < .05). There were no significant differences among the extenders for straight-line distance.     

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.     

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.     

An approach to rescue the fertility of stallions with a high level of hemospermia

A high amount of blood and not the mere presence of blood in equine semen impacts fertility. The aim of this study was to develop an approach to rescue the fertility of stallions with high hemospermia levels. Semen from 15 stallions was divided into four experimental groups: (a) Control—pure raw semen, (b) WB50—50% (v/v) whole blood added into semen, (c) E1—WB50 extended in a 1:1 (v/v) ratio with milk-based extender and (d) E2—WB50 extended in a 2:1 ratio with milk-based extender. Sperm kinetics, plasma membrane integrity (PMI), lipid peroxidation (PER) and intracellular superoxide (O₂) production were immediately evaluated. Four cycles of 20 mares were randomly assigned to the experimental groups. Mares were bred with an insemination dose of 1 billion total sperm and pregnancy was diagnosed 14 days after ovulation. Sperm kinetics could not be evaluated in the WB50 samples. Total motility was lower (p < .05) in E1 than in CT and E2 samples. Progressive motility decreased (p < .05) with an increase in the percentage of blood in the samples. The PMI and PER did not differ between groups (p > .05); however, O₂ production was higher (p < .05) in WB50 than in E2 samples, while the values were intermediate (p > .05) for CT and E1 samples. The control (90%) and E2 (90%) groups had superior (p < .05) fertility than the others (WB50—0% and E1—25%). It was concluded that sperm motility and fertility of semen with a large amount of blood can be rescued by dilution with a 2:1 extender:semen ratio using a milk-based extender.     

Effect of pyruvate and lactate on motility of cold stored stallion spermatozoa challenged by hydrogen peroxide

Numerous workers have shown that motility of cold stored spermatozoa from some stallions can be improved by removing most of the seminal plasma by centrifugation and resuspending the spermatozoa in an extender consisting of skim milk glucose extender (SKMG) supplemented with a salt media such as Tyrode's or phosphate-buffered saline. The salt media must contain pyruvate and lactate. In an effort to test the hypothesis that pyruvate may be acting as an antioxidant, a series of experiments were conducted using a H₂O₂ challenge to artificially produce damage due to lipid peroxidation. Results of these experiments indicated that addition of lactate or pyruvate and lactate to SKMG-Tyrode's media was not able to prevent the detrimental affects of H₂O₂. The addition of lactate to the SKMG-Tyrode's media resulted in an improvement of post-storage motility; however, increasing the concentration of pyruvate did not further improve motility. Therefore, because lactate dehydrogenase has been shown to be correlated with motility, and lactate has been shown to be preferred as an energy source by spermatozoa from other species, the beneficial effects of lactate and pyruvate as components of a modified SKMG extender are probable as energy sources.     

Influence of Steroidal Anti-Inflammatory Drugs on Viability and Fertility of Equine Semen

Considering the efficiency of steroidal anti-inflammatory drugs (SAIDs) in the immunomodulation of breeding-induced endometritis and the possibility of using these drugs by intrauterine route instead of the parenteral application, the present study aimed at evaluating the effect of SAIDs added to the semen extender on equine sperm viability and fertility. In experiment 1, 15 SAIDs were individually added to a skim milk-based extender and, based on the results of sperm motility, dexamethasone was the drug of choice for the subsequent trials. The effect of dexamethasone on the viability of fresh and 24-hour cooled semen was investigated in experiment 2. In experiment 3, fertility rate was measured in both post-breeding endometritis-resistant and susceptible mares. Although dexamethasone supplementation caused a premature decrease in sperm total and progressive motility and in sperm velocities (P < .05), no difference was observed for sperm membrane integrities and fertility (P > .05). Based on these results, we can conclude that dexamethasone can be added to equine semen at the time of insemination or before cooling, although its use was not able to increase fertility.     

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.     

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.     

Positive Effect of Manganese (III) Meso-Tetrakis (4-Benzoic Acid) Porphyrin on Stallion Spermatozoa During Storage in Cool Condition

The aim of the present study was to assess the effect of manganese (III) meso-tetrakis (4-benzoic acid) porphyrin (Mn-TBAP) on stallion sperm quality during storage at 5°C. In the present study, 18 ejaculates from three stallions were collected and diluted by INRA82 extender containing 0 (Mn-0), 100 (Mn-100), 200 (Mn-200), and 300 (Mn-300) μM of Mn-TBAP. Sperm motility, plasma membrane integrity and functionality, and lipid peroxidation as indicated by malondialdehyde (MDA) in the spermatozoa of diluted semen were evaluated in vitro at 2, 24, and 48 hours after storage at 5°C. The results showed that all evaluated sperm parameters, except MDA concentration, decreased significantly during the storage period. Total and progressive motility of spermatozoa were higher in Mn-200 extender (46.75 ± 0.58 and 27.62 ± 0.6, respectively) compared with Mn-0 (44.43 ± 0.58 and 25.13 ± 0.6, respectively) and Mn-300 (43.95 ± 0.58 and 25.28 ± 0.6, respectively) after 48 hours of storage at 5°C (P < .05). In addition, sperm plasma integrity and functionality were higher in Mn-200 extender (53.12 ± 0.6 and 46.63 ± 0.78, respectively) compared with Mn-0 (47.74 ± 0.6 and 40.96 ± 0.78, respectively), Mn-100 (48.63 ± 0.6 and 41.99 ± 078, respectively), and Mn-300 (46.11 ± 0.6 and 3.75 ± 0.78, respectively) after 48 hours of storage at 5°C (P < .05). The result showed also that MDA level was lower in Mn-100 extender (3.91 ± 0.06) compared with Mn-0 (4.51 ± 0.06), Mn-200 (4.25 ± 0.06), and Mn-300 (4.75 ± 0.06) after 48 hours of storage at 5°C (P < .05). In conclusion, INRA82 extender supplemented with 200-μM Mn-TBAP could efficiently preserve Caspian stallion spermatozoa after 48 hours of storage at 5°C.     

Addition of Glutathione to an Extender for Frozen Equine Semen

The manipulation of equine semen during cryopreservation reduces sperm viability and fertility because of, among other factors, membrane lipid peroxidation that makes cells highly susceptible to free radicals and reactive oxygen species (ROS). The oxidative effect caused by the generation of ROS can be reduced by the addition of antioxidants to the seminal plasma or to the extenders used for freezing. The current study was performed to test the in vitro effect of exogenous glutathione added in five different concentrations (control, 2.5 mM, 5.0 mM, 7.5 mM, and 10 mM [treatments 1-5, respectively]) to the extender for 12 stallions. Analyzed parameters were sperm motility, viability, and acrosome and plasmatic membrane integrity. Total motility was higher in treatments 1 and 2 (P < .05); viability, progressive motility, and plasmatic membrane integrity were higher in treatment 2 (P < .001). As for acrosome membrane integrity, treatment 3 showed the best results (P < .05). The addition of 2.5 mM glutathione to the freezing extender preserves total motility and increases sperm viability, progressive motility, and plasmatic membrane integrity. Concentrations above 2.5 mM were deleterious to spermatozoa.     

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.     

Water Absorption Capacity of Flax and Pine Horse Beddings and Gaseous Concentrations in Bedded Stalls

It can be a challenge to find suitable horse bedding materials that provide higher moisture absorption, better animal comfort, greater fertilizer values, and improved indoor environment. Our first objective was to determine the water absorption capacity (WAC) of two bedding materials, flax shive (FS) and pine wood shavings (PWS), commonly used by equine facilities. The second objective was to measure ammonia (NH₃), hydrogen sulfide (H₂S), and greenhouse gas (GHG) concentrations emitted from these bedded stall surfaces. In this study, the WAC of bedding materials were measured at 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 hours in the laboratory. A total of eight horses were used for a 14-day study period. Of these, four horses (group-1) were bedded with FS and the other four (group-2) were bedded with PWS for week-1. In week-2, bedding materials were switched between the two groups. Ammonia and H₂S were measured in situ. For GHG measurement, air samples (methane [CH₄], carbon dioxide [CO₂], and nitrous oxide [N₂O]) were collected 152 mm above the bedded stall surface in Tedlar bags using a vacuum chamber and analyzed for GHG using a gas chromatograph. The WAC of FS was 56% greater than the PWS. There were no significant differences in NH₃, H₂S, CH₄, CO₂, and N₂O concentrations between the two bedding materials (P > .05). Nutrient contents between fresh and soiled bedded samples for each bedding type were different (P < .05). Measured nutrient contents between fresh FS and PWS and bedded FS and PWS bedding materials were similar (P > .05).