Cryopreservation Of Stallion Semen Research Articles

Impact of the Antioxidant Hydroxytyrosol on the Quality of Post-Thawed Stallion Semen.

The objective of this study was to investigate the impact of including hydroxytyrosol (HT) antioxidant on the viability of sperm after the processes of cooling and freezing. HT antioxidants were used in the HF-20 extender at concentrations of 1.25, 2.5, 5, and 10 μg/ml. The HF-20 extender was a basic extender and was used for the control group. The post-thawed semen exhibited significantly higher total motility in the 2.5 HT and 10 HT treatment groups than the control group. The straight line velocity (VSL) of the 2.5 HT group exhibited a significantly high value compared with the control group. However, the average path velocity (VAP), linearity (LIN), straightness index (STR), and wobble (WOB) revealed identical findings across all groups. The findings of the analysis of HOST, normal morphology, major abnormalities, and minor abnormalities revealed that there were no significant differences between the HT groups and the control groups. Nevertheless, the use of HT antioxidant for freezing semen led to a notable enhancement (p < 0.05) in both acrosome integrity and vitality tests when compared to the control group. In this case, the lower quantities of HT (1.25 and 2.5 μg/ml; p < 0.05) preserve the DNA fragmentation compared with the 5 HT, 10 HT, and control groups. In conclusion, the HT antioxidant has shown the capacity to enhance the quality of frozen-thawed spermatozoa and positively influence the viability and integrity of DNA inside the frozen-thawed spermatozoa. Additional research should be conducted to assess the fertility potential of cryopreserved stallion semen.

Stallion semen cryopreservation: Frozen semen preparation, handling and breeding

SummaryThe use of cryopreserved stallion semen is a significant aspect of equine reproduction, despite the tendency for lower per cycle pregnancy rates than cooled or fresh semen. This decreased fertility of thawed frozen semen is due in part to the innate subfertility of the chosen male breeding population, but also to the direct insults to the sperm cell during the freezing and subsequent thawing process. The mare aspect of frozen semen breeding is intensively managed to compensate for the aforementioned damages, and acceptable pregnancy rates can be achieved. Here we describe the stallion semen cryopreservation process including semen collection, expected causes of damage in processing, freezing and thawing, mare management and future directions.

Effect of Extender Supplementation With Caffeine on Arabian Stallion’s Semen Quality After Freezing

In the current study, we aimed to investigate the effect of adding different levels of caffeine to the diluent on the Arabian stallion&amp;rsquo;s sperm quality post cryopreservation. Semen cryopreservation is an essential part of artificial insemination in Arabian horses. In most of cases, the stallion selection is based on desirable phenotypic and physical traits rather than on fertility. This approach may have resulted in the propagation of low-fertility genotypes that are unable to withstand the various stresses associated with cryopreservation such as freezing-thawing stress, oxidative stress, and osmotic stress. Caffeine has been shown to act as a phosphodiesterase inhibitor which may increase the level of cAMP in sperm and subsequently, increase the formation of adenosine triphosphate, which is important for sperm motility. Because it contains xanthine and theobromine, caffeine has antioxidant properties that increase sperm motility and their fertilizability. Five healthy stallions aged 4 to 10 years have been used for semen collection. Semen collections has been done twice weekly by artificial vagina. The ejaculates were evaluated for general progressive motility and sperm concentration. Sperm concentration and motility were determined using the CASA system (ISAS program, Prosser R+D, Paterna, Valencia, Spain). Samples with a minimum concentration of 200 &amp;times; 106 sperm/ml and motility &amp;gt; 60% used for this study. The filtered semen of each ejaculate was diluted (1:1) and divided into 4 aliquots. The aliquots were centrifuged at 800 g for 10 minutes, the seminal plasma was removed, and each sample was re-suspended with FH-20 (0) without any supplementation as a control or with caffeine at a concentration of 0.5 mM, 1 mM, 3 mM or 5 mM. The final semen concentration after dilution was 200 &amp;times; 106 sperm/ml. All samples were assessed before freezing in liquid nitrogen. The frozen thawed semen was assessed for general and progressive motility and then evaluated using the ISAS program and assessed for plasma membrane integrity, morphology defects, acrosome integrity, and viability. Descriptive analyses were determined for the evaluated variables. Statistical comparisons between groups were performed with one-way analysis of variance (ANOVA) (p &amp;lt; 0.05). The addition of 1 mM caffeine to the extender gave better results in total motility (TM), progressive motility (PM) and vitality. However, all studied caffeine concentrations had good effects on straight line velocity (VSL), straightness (STR), linearity (LIN), wobble (WOB) and HOST with no effects on acrosome integrity. It could be concluded that caffeine has a beneficial effect on Arabian stallion semen cryopreservation when added at a concentration of 1 mM to the extender.

Optimization of protocol for stallion semen cryopreservation

The aim of the preliminary study was to improve success of cryopreservation of stallion spermatozoa. The effect of different equilibration lengths and two techniques for freezing of stallion semen was evaluated, based on motility and viability parameters. Fresh semen was diluted with BotuCrio extender and divided into 3 groups equilibrated for 0.5h, 2h, and 4h. Subsequently, half straws of each group were cryopreserved in the vapours of the liquid nitrogen or in the automatic freezer. Results of stallion spermatozoa motility and progressive motility showed decreasing trend with increased time of equilibration. Moreover, a significant difference (p &lt; 0.05) for the percentage of spermatozoa producing ROS was found between semen frozen in the vapours of the liquid nitrogen compared to semen cryopreserved by the automatic freezer in the same equilibration time. Nevertheless, results of this study are of preliminary character. Experiments using higher number of individuals need to be tested in order to find the best procedure for semen cryopreservation of Slovak national horse breeds.

Novel lyophilized extenders maintain stallion semen characteristics during freezing but do not need frozen shipment or storage

Stallion semen cryopreservation is an important procedure for conservation of genetic resources having a relevant roll in the horse industry. Distribution of commercial extenders is difficult due to the necessity of keeping them frozen during transport. The aim of this work was to compare after-thawing results of two commercial frozen extenders offered worldwide (C1 and C2) with two novel lyophilized freezing extenders produced in Brazil (STAR and MX3) which can be stored at room temperature up to one year. One ejaculate of each of 28 mature stallions from three breeds (Quarter Horse, Criollo and Mangalarga Marchador) was diluted with milk-based extender (Equiplus, Minitube) to 100 × 106 total sperm/ml and split into four 50ml centrifugation tubes. After centrifugation (600 g x 20’), the remaining pellet of each tube was resuspended with one of the four extenders to a concentration of 200 × 106 total sperm/ml. From each aliquot, semen was frozen with two different freezing curves named FC1 (Room temperature + 20’ at 5˚C into refrigerator + 20’ followed by LN2 vapor and plunging) and FC2 (60’ at 5˚C). After freezing, straws were thawed at 37˚C/30 sec and total motility (TM) was checked using optical microscopy in a blind test by three skilled vets. Means± standard deviation for total motility after freezing-thawing were: FC1 (C1=46.54±19.44;C2=35.30±21.32; STAR=51.15±21.18 and MX3=46.17±20.50) and FC2 (C1=51.55±21.92; C2=41.61±19.79; STAR=44.85±16.30 and MX3=49.21±15.98). Statistical analysis was performed (ANOVA followed by Tukey test) with 5% significance. No differences on average motility among extenders within or between curves was seen. Within each curve, the distribution of best motilities of each extender considering individual stallions was FC1 (C1=6, C2=2, STAR=14, MX3=6) and FC2 (C1=14, C2=4, STAR=4, MX3=6). The results show similarity on average motility for all extenders tested. Considering FC1, the lyophilized extenders got better motility in 20 of 28 stallions. In FC2, C1 performed better (14 of 28 stallions), while STAR and MX3 were comparable with the commercial extender C2. MX3 had similar performance in both curves and STAR reached better thawed motility in FC1 (14 of 28 stallions) when compared with FC2 (4 of 28 stallions). The highest motility obtained after the freezing-test revealed a balance between FC1 and FC2 with 13 and 15 stallions, respectively. Individual variation and freezing curve splitting were similar for both kinds of extenders, but further investigations are needed to clarify the better results of STAR when using faster curves. In conclusion, lyophilized extenders can be a good option to obtain similar semen quality without restrictions of frozen extender shipment and storage.

The effect of residual pellet volume on cryoprotectant concentration and post-thaw motility of stallion sperm

Standard protocols for cryopreservation of stallion sperm involve centrifugation to remove plasma and concentrate ejaculates. Typically, semen is diluted 1:1 or higher to a concentration of 50 to 100 × 106/ml and centrifuged on a cushion. Most supernatant and cushion is aspirated leaving 5-10% of the original volume of diluted semen along with the sperm pellet. Pellets are resuspended in freezing diluent containing cryoprotectant to 200 × 106 sperm/ml. Due to the extreme variability of sperm concentration between ejaculates, the volume of resuspended sperm pellet and supernatant may contribute significantly to the final volume of the extended semen resulting in variability in the final percent of cryoprotectant (CPA) in the extended semen. To determine the degree of variability and the effect of the final CPA concentration on retention of sperm motility we determined final CPA concentration in extended semen of 129 ejaculates from 16 different stallions. Our hypothesis was that ejaculates cryopreserved with CPA concentration significantly lower than that present in the freezing diluent would have lower cryosurvival. Pellet volume was determined by weight and used to calculate the final CPA concentration prior to freezing for each ejaculate. The final CPA concentration was expressed as percentage of the CPA concentration in the original freezing extender. For the 129 ejaculates, the mean final CPA concentration aspercentage of original was 69.5%, median 73.7%, min 39.2% and max 87.8%. Final CPA concentration 1.3 - 5.1%. Sperm motility was analyzed by CASA for fresh extended and post-thaw samples. Thawed samples were diluted in centrifugation media and incubated (37C for 30 minutes) prior to analysis. For each sample a minimum of 5 fields and 400 motile sperm were analyzed (40 frames at 60 frames/sec); progressive cells defined as (VAP > 50 mic/sec and STR > 75%). Cryosurvival was expressed as percentage change in motility between pre-freeze and post-thaw values. Ejaculates were divided into 5 groups based on final CPA concentration as percentage of the original extender amount (G1 = 40-50%, G2 = 50-60%, G3 = 60-70%, G4 = 70-80%, G5 = 80-90%). Mean values for change in motility were compared between groups using ANOVA with multiple comparisons. Decrease in percent total motility (mean,sd) was less (p<0.05) in G5 (22.8,11.0) compared to all other groups G4 (31.3,11.3), G3 (31.2,13.1), G2 (29.5,8.8), G1 (30.9,10.8). Decrease in progressive motility was less (p<0.05) in G4 (8.5,14.8) and G5 (2.7,10.5) vs G1(17.8,8.7), G5 vs G2 (13.6,10.9) and G5 vs G3 (12.9,14.3). These data suggest that current protocols for cryopreservation of stallion semen may result in inadequate CPA concentration in extended semen of many ejaculates. Further experiments are underway to determine critical thresholds.

Freezability and Fertility Rates of Stallion Semen Supplemented With Trehalose in Lactose Extender

Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. One of the reason for this diminished quality is osmotic stress that spermatozoa experiences during freezing and thawing process. 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–thawing process. Semen samples were collected from six Marwari breed stallions, divided into three different treatments in a final concentration of 150 × 106 sperm/mL by using Lactose based extender containing 0, 50, and 150 mM of trehalose then subjected to cryopreservation after equilibration. Sperm motility, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, DNA integrity and oxidative stress related parameters of the stallion spermatozoa were analyzed at fresh, prefreeze and post thaw stages. Thirty (30) reproductively healthy mares were inseminated with frozen-thawed semen either supplemented with (treatment) or without (control) trehalose to evaluate the field fertility. Results of the current study indicated that, the extender containing 50 mM trehalose has enhanced the functional plasma membrane, acrosomal, DNA integrities and augmented the mitochondrial membrane potential. Trehalose supplementation to the semen extender not only ameliorated the semen quality parameters, but also protected the stallion sperm from oxidative stress by reducing the levels of reactive oxygen species (ROS) and lipid peroxidation (LPO). The inclusion of 50 mM trehalose in semen extender resulted in significantly (P < .05) increased post-thaw progressive motility and viability compared to the control group. Mares inseminated with frozen-thawed semen supplemented with 50 mM trehalose tended to have better pregnancy rates than controls (non-significant [P < .05]) although a larger fertility trial is required to determine if this effect reaches the level of significance. In conclusion, addition of 50 mM trehalose yielded in better quality stallion semen after cooling and post-thawing in terms of reducing the oxidative stress and enhancing the motility, integrities of acrosome, plasma membrane, mitochondrial potential and DNA.

Asthenozoospermic stallions tend to have high acrosome reacted spermatozoa as evidenced by dual fluorescent staining assay

Acrosome intactness of spermatozoa is the critical factor for establishing sperm reservoir in oviduct and for fertilizing an oocyte. However, frozen thawed spermatozoa tend to show higher proportion of acrosome reacted spermatozoa thereby compromising the fertility. Conventional staining techniques identify only sperm acrosome integrity and not precisely the acrosome reaction status. In this context, the current study was conducted to assess the acrosome status of cryopreserved spermatozoa using Fluorescein isothiocyanate conjugated peanut agglutinin and propidium iodide (FITC-PNA+PI) in stallions with varying sperm motility. Stallions were classified into high- (≥45%) and low-motile group (≤30%) based on their post-thaw sperm motility. The proportion of live acrosome intact (LAI) spermatozoa was significantly higher in high-motile group as compared to low-motile group. A significant positive correlation was observed between LAI and post-thaw sperm motility. In conclusion, the present study showed that FITC-PNA+PI combination could be used for rapid and accurate assessment of acrosome reaction status of stallion spermatozoa, and the proportion of LAI population in cryopreserved stallion semen had a strong correlation with sperm motility.

Effect of cholestanol and cholesterol-loaded cyclodextrin on stallion sperm function and capacitation post-cryopreservation

Cryopreservation of stallion semen is less efficient than other species such as bovine. This is mainly because of the greater susceptibility of stallion sperm to the freezing damage that generates oxidative stress and plasma membrane injury, resulting in DNA fragmentation and cell death.These data suggest the need to develop new strategies of sperm cryopreservation that can improve the efficiency of this technique in stallions by reducing or preventing membrane damage and cell death. The present study aimed to evaluate the effect of adding membrane stabilizers to the freezing medium and assess the quality and in vitro capacitation of stallion sperm after thawing. Semen samples from three stallions frozen with membrane stabilizers (cholesterol-loaded cyclodextrin and cholestanol-loaded cyclodextrin) were evaluated in two experiments: i) sperm quality and functional analysis after thawing, and ii) sperm quality and functional analysis after 4 h of post-thaw incubation in capacitating conditions. Plasma membrane integrity, mitochondrial membrane potential, membrane lipid disorder, intracellular Ca2+, tyrosine phosphorylation, acrosome reaction, DNA damage, sperm motility, and binding to the zona pellucida were assessed. The results showed that cholesterol-loaded cyclodextrin was the stabilizer that most efficiently reduced the membrane disruption and post-thaw cell damage. In addition, this stabilizer made it possible to obtain in vitro capacitated sperm showing higher plasma membrane integrity, mitochondrial membrane potential, sperm motility, binding to the zona pellucida and better response to in vitro capacitating conditions.

Quality and Fertility of Trehalose Supplemented Cryopreserved Stallion Semen

Quality and Fertility of Trehalose Supplemented Cryopreserved Stallion Semen

Effects of Spermine and Spermidine supplemented extenders on post-thaw Spermatological Parameters in Stallion Semen Cryopreservation

In this study, the effects of polyamines, Spermine and Spermidine, on long-term preservation and post-thaw spermatological parameters were evaluated. Moreover, determination of the most suitable polyamine and its dose that can be added to standard extenders were aimed. Four adult Arabian stallions were used in the study. Five ejaculates were collected from each of four stallions via artificial vagina two days interval. Each ejaculate was divided into 13 aliquots. INRA96 (95,5%), egg yolk (2%), and glycerol (2,5%) were used as a control extender. Extenders of experimental groups were prepared with different doses of Spermine and Spermidine (0,1 mg/ml; 0,2 mg/ml; 0,4 mg/ml; 1 mg/ml; 2 mg/ml; 4 mg/ml). Stallion semen that were cryopreserved with Control and experimental extenders were evaluated in terms of Total Motility, Progressive Motility, Plasma Membrane Integrity, Capacitation Index, Acrosome Integrity and DNA Fragmentation Index. At the end of the evaluations, it was determined that 0,2 mg/ml Spermine and 0,4 mg/ml Spermidine showed better Total Motility and Progressive Motility, numerically. On the other hand, it was observed that 4 mg/ml Spermine and Spermidine had the lowest statistically significant values (p < 0,001). While statistically similar differences were obtained between groups in terms of the Plasma Membrane and Acrosome Integrity, it was determined that all experimental groups had lower and statistically significant values in terms of Capacitation and DNA Fragmentation Index (p < 0,001). As result, it was observed that the stallion semen cryopreservation success can be increased by the addition of 1–2 mg/ml Spermine that had effective protection on Capacitation and DNA Fragmentation Index without damaging other spermatological properties.

Cryopreservation of stallion semen: Effect of adding antioxidants to the freezing medium on sperm physiology.

Cryopreservation of stallion semen has not reached the level of efficiency and positive results described in other species. This is mainly due to the greater sensitivity of stallion sperm to the freezing process, showing higher rates of oxidative stress and plasma membrane damage, which trigger the activation of several cell damage pathways that ultimately culminate in DNA fragmentation and cell death. Therefore, finding molecules that improve the efficiency of this technique in stallion by preventing oxidative stress and cell damage is required. Thus, the aim of the present study was to evaluate the effect of adding three antioxidants (MnTBAP, NAC and FeTPPS) to the freezing medium on the quality and functional parameters of stallion sperm. Semen samples from three stallions frozen with the antioxidants were evaluated in two conditions: (a) adding the antioxidants before freezing, and (b) before and after freezing. Plasma membrane integrity, mitochondrial membrane potential, lipid peroxidation, intracellular ROS levels, membrane lipid disorder, DNA damage, sperm motility and binding to the zona pellucida were assessed. The results showed that MnTBAP was the antioxidant treatment that best controlled the oxidative stress process and post-thaw cell damage, showing higher plasma membrane integrity, mitochondrial membrane potential, sperm motility, number of spermatozoa bound to the zona pellucida of bovine oocytes and lower lipid disorder. Additionally, it was determined that a second post-thaw application of antioxidants is detrimental since induced higher cell damage and lower sperm motility, without showing any beneficial effect on the spermatozoa.

In vitro addition of DHA and IGF-I increases the progressive motility of cryopreserved stallion semen

High seminal quality is important to achieve acceptable pregnancy rate following artificial insemination with cryopreserved semen in the equine industry. Docosahexaenoic acid (DHA) is an omega-3-polyunsaturated acid, which improves the integrity of the spermatozoa membrane during temperature changes. Insulin-like growth factor-I (IGF-I) is a protein hormone that helps mainly glucose to enter spermatozoa and it is an antioxidant. The aim of this study was to assess the effect of the in vitro addition of DHA in combination with IGF-I to frozen-thawed stallion semen. Three ejaculates from each of three Irish Sport Horse stallions were collected, the gel fraction was removed and semen was diluted in a 1:1 ratio using extender, centrifuged (1.000 g) for 10 minutes at 32 °C and ressuspended to 100 x 106 spermatozoa/mL in freezing extender. Semen was cooled to 4 °C, packed into 0.5 mL straws, frozen and stored under liquid nitrogen at -196 °C. Straws were thawed at 37 °C for 30 seconds, semen was diluted to 25 x 106 spermatozoa/mL and split in four treatments adding 0 or 1 ng of DHA /mL and 0 or 100 ng of IGF-I /mL: DHA0, DHA0 + IGF-I, DHA1 and DHA1 + IGF-I. Semen was incubated at 32 °C and after 30 minutes, total motility (TM), rapid progressive motility (PM), viability and acrosome integrity were assessed. After 60 and 120 minutes, TM and PM were assessed again. Post-thawed PM was higher (P &lt; 0.05) when DHA1 + IGF-I was added, but there was no effect of the addition of DHA and IGF-I to TM, viability or acrosome integrity (P &gt; 0.05). Keywords : Equine. Spermatozoa. Omega-3. Antioxidant. Hormone.

Acute Endometritis due to Taylorella equigenitalis Transmission by Insemination of Cryopreserved Stallion Semen

Taylorella equigenitalis can be transmitted during artificial insemination. This report describes clinical T. equigenitalis transmission by cryopreserved stallion semen. T. equigenitalis isolates from a mare's vaginal discharge and semen from the same batch of the cryopreserved semen used for the insemination gave identical API ZYM, antibiotic susceptibility, and multilocus sequence typing results (ST-46); furthermore, the multilocus sequence typing lineage ST-46 is known to circulate in the country of semen collection. These results support the need for strict contagious equine metritis screening of processed semen before use for artificial insemination.

Postthaw Addition of Autologous Seminal Plasma Improves Sperm Motion Characteristics in Fair and Poor Freezer Stallions

During semen processing for cryopreservation, most seminal plasma is usually removed, and components with protective effects on sperm may be missing after thawing and within the female reproductive tract. The present study evaluated the effect of postthaw addition of autologous seminal plasma on motion characteristics of stallion sperm with fair (n = 4) or poor (n = 3) freezability. Therefore, pure seminal plasma (group SP1), seminal plasma combined with fresh semen extender (group SP2), or seminal plasma mixed with freezing extender (group SP3) were used to fill 0.5 mL straws and frozen similar to stallion semen. Postthawing, semen samples (n = 42) were diluted either with semen extender (group FT) or with seminal plasma (n = 126) of groups SP1 to SP3 to 25 × 106 sperm/mL. In fair freezer stallions, total and progressive motilities were higher in group FT than in group SP1 (P < .05), but there was no difference in poor freezing stallions among groups (P > .05). However, comparing individual stallions, positive effects of seminal plasma on total or progressive motility were detected in two stallions. Curvilinear velocity increased in groups SP2 and SP3 in fair freezer stallions and in all groups with seminal plasma compared with group FT in poor freezer stallions (P < .05). Although straightness was higher in groups SP2 and SP3 compared with group FT in fair freezer stallions (P < .05), there was no difference among groups in stallions with poor freezability (P > .05). Average lateral head displacement did not change among groups of fair freezer stallions (P > .05) but was higher in groups SP2 and SP3 than in group FT in poor freezer stallions (P < .05). Beat cross frequency was higher in all groups diluted with seminal plasma postthawing in fair freezer stallions (P < .05), but only in group SP1 than in group FT in poor freezer stallions (P < .05). The addition of autologous seminal plasma to frozen-thawed semen can improve motion characteristics of stallions with fair and poor freezability. This is a valuable additional protocol for laboratories dealing with cryopreservation of stallion semen and for veterinarians working with fair or poor freezer stallions.

Effect of Using Two Cryopreservation Methods on Viability and Fertility of Frozen Stallion Sperm

Studies involving different methods and techniques of cryopreservation and its interactions with the conception rates in artificial insemination (AI) programs are reported in the literature. This study evaluated the sperm kinetics, plasma membrane integrity, and fertility rates of mares inseminated with cryopreserved stallion semen subjected to different freezing methods. For this, four ejaculates from five stallions were collected and frozen in conventional (Styrofoam box) or automated system in Mini-Digitcool ZH 400. Seminal samples were evaluated after thawing for sperm motion parameters by CASA and plasma membrane integrity by epifluorescence microscopy. For the fertility trial, a cross-over model was performed using 100 cycles of 50 mares, which were inseminated by one the two freezing methods. No differences were observed for sperm motion parameters and plasma membrane integrity between groups (P > .05). The pregnancy rate using the conventional method was 56% (28/50) and did not differ (P = .5406) from the pregnancy rate (64%, 32/50) obtained using the automatized method. The use of semen from fertile stallions may not illustrate small differences in the two freezing methods evaluated. Conventional and automated freezing systems did not differ in the quality and viability of fertile stallion semen and conception rates, indicating that the two methodologies can be safely used in AI programs.

Using Resveratrol and Epigallocatechin-3-Gallate to Improve Cryopreservation of Stallion Spermatozoa With Low Quality

Reactive oxygen species levels in semen with low quality increased from 25% to about 40%. Resveratrol (Res) is a natural grape-derived phytoalexin, and epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis); both molecules are known to have high levels of antioxidant activity. The aim of this study was to investigate the effects of this antioxidant's supplementation on cryopreservation of stallion semen with poor quality. Experiment 1: Experiment design to asses stallions semen quality (n = 3). Arabian stallions were managed for breeding with fresh, extended, or chilled semen during 2 years (in two breeding season) with a resulting per cycle pregnancy rate (103/41 estrus cycle/conception) of 39.80%. A total of 81 ejaculates were collected and evaluated in fresh condition (total and progressive motility, membrane integrity, acrosome defects, morphology, and abnormal DNA condensation). Evaluation of several ejaculates revealed that sperm production and semen quality mostly did not change during the breeding season, maintaining normal sperm production with poor quality. Experiment 2: 18 ejaculates, collected with Missouri model artificial vagina from Arabian stallion (n = 3). Extended semen was centrifuged at 600 × g for 10 minutes. To eliminate individual stallion variability, the resulting sperm pellets were pooled and divided in seven different treatments in a final concentration of 100 × 106 sperm/mL by using semiskimmed milk extender containing no supplements (control), and the following supplements with three different concentrations: Res 5, 10, and 20 μM (Res 1, Res 2, Res 3, respectively) and EGCG 25, 50, 100 μM (EGCG 1, EGCG 2, and EGCG 3, respectively). After thawing, all samples were maintained at 37°C for 10 minutes. Evaluation of motility, plasma membrane integrity, viability, mitochondria activity, DNA fragmentation, and lipid peroxidation was determined. For all semen parameters, the lower concentration of Res (5 μM) and EGCG (25 μM) did not show significant differences compared to control (P > .05). Relative to the control group, Res 2 had better (P < .05) postthaw total and progressive motility, yet the higher concentrations (Res 3) showed a negative effect on total motility. Percentage of membrane integrity, viability, and mitochondrial activity was significantly greater in Res 2 and EGCG 2 extenders in comparison with the control group, respectively (P < .05). Results showed that malondialdehyde concentration was lower in Res 2 and EGCG 2 groups. In conclusion, the present study showed that the antioxidant Res prevents DNA damages in our stallions. The addition of Res and EGCG before cryopreservation can ameliorate mitochondrial activity in stallions with low-quality semen. Although further studies are needed, the present work showed that Res and EGCG could be considered as cryopreservation procedures in stallions with low-quality sperm and possibly equine, to avoid or minimize DNA damages, and preserve sperm plasma membrane integrity and mitochondrial activity.

Intracellular calcium chelating agent (BAPTA-AM) aids stallion semen cooling and freezing-thawing.

This study aimed to investigate the effects of different concentrations of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N0N0-tetraacetic acid, tetra-acetoxymethyl ester (BAPTA-AM), an intracellular calcium chelating agent, on stallion semen cooling and freezing-thawing. After collection, semen was extended (1:1 v/v) on a skim milk-based extender, centrifuged and resuspended at 400million/ml into cooling or freezing extenders containing 0, 5, 25, 50, 100 and 200μΜ BAPTA-AM. Motility parameters were assessed after cooling in Equitainer at 5°C for 12, 24, 48, 72 and 120hr and after freezing-thawing. In addition, mitochondrial membrane potential, intracellular ATP, reactive oxygen species and malondialdehyde concentrations were measured in cryopreserved-thawed semen. Cooled stored (48hr) semen containing 50μΜ BAPTA-AM and control extender (0μΜ BAPTA-AM) was used to assess fertility. Inclusion of 50μΜ BAPTA-AM resulted in superior sperm motility parameters during cooled storage when compared to other groups (p<0.05). Furthermore, semen cryopreserved in extender containing 50μΜ BAPTA-AM showed increased intracellular ATP and mitochondrial membrane potential, whereas reactive oxygen species and malondialdehyde were increased after thawing for all groups (p<0.05). Addition of 50μΜ BAPTA-AM to cooling extender resulted in similar pregnancy rates to the control group (75% vs. 73.6%, respectively; p>0.05). In conclusion, the addition of BAPTA-AM to semen extenders aided stallion semen cryopreservation in a dose-dependent manner. Furthermore, the cooling extender supplemented with 50μΜ BAPTA-AM could be used to prolong the sperm motility during cooling without apparently compromising fertility. Field trials should be conducted to assess fertility of cryopreserved stallion semen with BAPTA-AM.

Superoxide Anion Is Reduced in Gradient Selected Cryopreserved Stallion Semen Despite High Mitochondrial Potential

Superoxide Anion Is Reduced in Gradient Selected Cryopreserved Stallion Semen Despite High Mitochondrial Potential

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.