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Simple Summary: Freezing stallion semen yields variable results, heavily dependent on individual differences, categorizing stallions as good or poor freezers. This variability was investigated by analyzing sperm kinetics, mitochondrial membrane potential, and hydrogen peroxide content in sperm samples treated with different extenders and procedures. Additionally, antioxidant activities based on ABTS and FRAP assays, along with nitric oxide stable metabolites (NOx), were assessed in seminal plasma, blank extenders, and extenders conditioned by spermatozoa either before or after freezing. Strong individual variability was observed in most sperm functions both before and after freezing. Several physical and chemical differences were noted between the shipping and freezing extenders. However, no significant differences in sperm function, antioxidant activities, and NOx values were found among the shipping extenders. In contrast, significant differences in antioxidant activities and NOx values were found between the freezing extenders. The significant interaction between stallion and extender suggests that customizing freezing procedures can improve sperm freezing outcomes in stallions. Sensitivity to freezing remains a critical issue in stallion semen cryopreservation procedures. To explore this topic in-depth, semen was collected from ten stallions, diluted with three different extenders, transported to the laboratory, and then centrifuged and frozen with four different extenders. We conducted analyses of sperm kinetics, mitochondrial membrane potential (MMP), and hydrogen peroxide content both before and after freezing. Additionally, we assessed antioxidant activity using the ABTS and FRAP methods and measured nitric oxide stable metabolites (NOx) in the blank extenders, seminal plasma, and extenders conditioned by spermatozoa before and after freezing. We found significant variability in the antioxidant activity and NOx content of the blank extenders and the seminal plasma. In the seminal plasma, ABTS-based antioxidant activity and NOx values were correlated with some sperm kinematic parameters and MMP in refrigerated semen, while no correlation was observed in frozen sperm parameters. Sperm function varied significantly between stallions but not between extenders, either before or after freezing. However, significant differences in antioxidant activities and NOx values were found among extenders conditioned following freezing. These results provide new insights into the factors contributing to the variability in individual stallions' tolerance to sperm freezing. [ABSTRACT FROM AUTHOR]

Summary: The 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. [ABSTRACT FROM AUTHOR]

Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mainly overrepresented in the "metabolism" and the "metabolism of lipids" pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays revealed that sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of differential abundance proteins, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In the current study, we examined: 1) the agreement (bias) between fluorescence-based methods (NucleoCounter-SP100 [NC] vs. flow cytometry [FC]) for determining the viability (VIAB) of frozen/thawed stallion sperm; 2) the agreement between post-thaw sperm total motility (TMOT) and VIAB; 3) whether a difference between TMOT and VIAB [VIAB – TMOT] in frozen/thawed stallion sperm could be explained by the level of lipid peroxidation in viable sperm (VLPP); 4) the repeatability of post-thaw analysis of sperm quality; and 5) the effect of final post-thaw semen dilution (10, 30, or 50 x 106 sperm/mL) on sperm motion characteristics. Post-thaw VIAB was similar between NC and FC (P > 0.05), and the agreement between these two methods was high (bias : 1 to −3). The agreement between post-thaw TMOT and VIAB decreased as the pre-freeze percentages of morphologically normal sperm and DNA quality decreased: bias – 4 to – 25. The bias between [VIAB – TMOT] and VLPP ranged from – 5 to 7. Differences in post-thaw sperm quality (TMOT, PMOT, VIAB, and sperm concentration) were not observed when analyzing one or three straws per ejaculate (P > 0.05). There was no effect of post-thaw sperm concentration (i.e., 10 vs. 30 vs. 50 x 106 sperm/mL) on sperm motion characteristics (P > 0.05). This study reports factors other than post-thaw sperm motility that warrant further consideration when analyzing frozen/thawed stallion sperm. • Post-thaw sperm viability was similar between NucleoCounter and Flow Cytometry across multiple levels of measurement. • The agreement between post-thaw motility and viability decreased as the intrinsic sperm quality decreased. • Lipid peroxidation was associated with reduced motility in the face of high viability in fresh but not frozen/thawed sperm. • Analysis of one straw per ejaculate yielded similar results than analyzing three straws per ejaculate. • Increasing the sperm concentration in thawed semen prior to motility analysis did not affect the CASA outcomes. [ABSTRACT FROM AUTHOR]

Scrotal surface thermography is a non-invasive method for assessing testicular thermoregulation in stallions; however, few studies have explored the application of this technique concerning the thermal physiology of equine reproductive systems. This study aimed to evaluate the consistency of testicular thermoregulation in stallions over a year using thermography to measure the scrotal surface temperature (SST). Moreover, we assessed the best region for measuring the surface body temperature compared with the SST. Ten light-breed stallions were used in the experiment. Thermographic images of the scrotal and body surfaces (neck and abdomen) were captured. Fresh, cooled and frozen-thawed semen samples were evaluated to verify the impact of thermoregulation on semen quality. Testicular thermoregulation was maintained throughout the year in stallions amidst changes in the external temperature, as evidenced by the weak correlation between the SST and ambient temperature. A lower correlation was observed between the environmental temperature and body surface temperature (BTS) obtained from the abdomen (BTS-A; R = .4772; p < .0001) than with that obtained from the neck (BTS-N; R = .7259; p < .0001). Moreover, both BTS-A and SST were simultaneously captured in a single image. The consistent quality of the fresh, cooled and frozen semen suggests efficient thermoregulation in stallions throughout the year. [ABSTRACT FROM AUTHOR]

Subfertility is one of the main issues in horse breeding and the study of mRNAs in sperm might help in elucidating the reasons that lead to this diagnosis. The present study aims at assessing the differences in the expression of 10 potential candidate genes in stallions of different fertility. Frozen-thawed semen of 29 stallions was included. Each sample was classified into two groups according to pregnancy rates (PR) achieved with this semen: "good fertility" (GF; n = 17; PR ≥ 30 %) or "poor fertility" (PF; n = 12; PR <20 %). All stallions underwent a breeding soundness examination (BSE) before semen production and were only included into the semen cryopreservation program when raw semen characteristics at BSE met minimal requirements. Semen was cryopreserved following European Union regulations and all stallions met the respective health requirements. Each sample was assessed for concentration (NucleoCounter SP-100), motility (CASA), membrane functionality (SYBR-14/PI), mitochondrial membrane potential (JC-1), morphology (SpermacStain), acrosome integrity (SpermacStain), membrane integrity (HOS test) and chromatin integrity (Aniline blue). Sperm RNAs were extracted using the Direct-zol RNA Miniprep Kit (Zymo Research) and RT-qPCR was performed for each target gene. ACTB and RPL32 were included as reference genes (RGs) for normalization. For each variable of each group, mean, standard deviation and SEM were calculated. The difference in gene expression levels between the GF and PF group were analyzed using the Mann-Whitney U test and Spearman's rank correlation. Significant results were considered with p < 0.05. Sperm quality parameters did not differ significantly between the two groups except for concentration, that was significantly higher in GF (p = 0.043). In GF a positive correlation was identified for PRM1 / PRM2 with r = +0.6, while PRM1 / ACR (r = −0.495), PRM2 / ZPBP (r = −0.645) and CRISP3 / ACR (r = −0.551) were inversely correlated. In PF direct correlations were registered for PRM1 / PRM2 (r = +0.629), PRM1 / PRM3 (r = +0.657), PRM2 / SPA17 (r = +0.685), SPA17 / PLCZ1 (r = +0.786) and PRM3 / ACR (r = +0.627). In the total sample (GF + PF), positive correlations were detected for PRM1 / PRM2 (r = +0.625), PRM1 / PRM3 (r = +0.368); PRM2 / SPA17 (r = +0.465), SPA17 / PLCZ1 (r = +0.637) and PLCZ1 / ZAN (r = +0.587). Only two of the genes considered were differentially expressed in the 2 groups: PRM2 and PLCZ1 , that were significantly (p < 0.05) overexpressed in the GF group. Stallions frozen-thawed semen with higher expression levels of PRM2 and PLCZ1 are more likely to belong to animals with a good pregnancy rate. Further studies are needed to investigate the role of sperm transcripts in male subfertility in stallions. • Sperm concentration was increased in frozen-thawed stallion semen with good fertility (GF). • The mRNA abundance of protamine 2 (PRM2) was increased in GF semen. • The mRNA abundance of phospholipase C zeta 1 (PLCZ1) was increased in GF semen. • Analysis of PRM2 and PLCZ1 mRNA abundance helps to predict frozen semen fertility. [ABSTRACT FROM AUTHOR]

If a mechanism of more efficient glycolysis depending on pyruvate is present in stallion spermatozoa, detrimental effects of higher glucose concentrations that are common in current commercial extenders could be counteracted. To test this hypothesis, spermatozoa were incubated in a 67 mM Glucose modified Tyrode's media in the presence of 1- or 10-mM pyruvate and in the Tyrode's basal media which contains 5 mM glucose. Spermatozoa incubated for 3 h at 37 °C in 67 mM Tyrode's media with 10 mM pyruvate showed increased motility in comparison with aliquots incubated in Tyrode's 5 mM glucose and Tyrode's 67 mM glucose (57.1 ± 3.5 and 58.1 ± 1.9 to 73.0 ± 1.1 %; P < 0.01). Spermatozoa incubated in Tyrode's with 67 mM glucose 10 mM pyruvate maintained the viability along the incubation (64.03 ± 15.4 vs 61.3 ± 10.2), while spermatozoa incubated in 67 mM Glucose-Tyrode's showed a decrease in viability (38.01 ± 11.2, P < 0.01). 40 mM oxamate, an inhibitor of the lactate dehydrogenase LDH, reduced sperm viability (P < 0.05, from 76 ± 5 in 67 mM Glucose/10 mM pyruvate to 68.0 ± 4.3 %, P < 0.05). Apoptotic markers increased in the presence of oxamate. (P < 0.01). UHPLC/MS/MS showed that 10 mM pyruvate increased pyruvate, lactate, ATP and NAD+ while phosphoenolpyruvate decreased. The mechanisms that explain the improvement of in presence of 10 mM pyruvate involve the conversion of lactate to pyruvate and increased NAD+ enhancing the efficiency of the glycolysis. • 10 mM pyruvate in a 67 mM Glucose-Tyrode's media maintains sperm functionality in comparison with a 67 mM glucose- Tyrode's. • Motility, linear motility, velocities membrane integrity, mitochondrial membrane potential and glutathione content were higher in the 10 mM pyruvate, 67 mM glucose media. • LACTATE and NAD+ content were higher in the spermatozoa incubated in the 10 mM pyruvate in a 67 mM Glucose-Tyrode's media. • The mechanism explaining the improvements may involve conversion of pyruvate to lactate and regeneration of NAD+. • The stallion spermatozoa may produce significant amounts of lactate under high Oxygen (Warburg effect). [ABSTRACT FROM AUTHOR]

Simple Summary: Most artificial inseminations in horses currently use cooled semen. It has not been possible to develop a freezing protocol that is suitable for all stallions and all ejaculates. Furthermore, the thawed spermatozoa have a short life, which necessitates depositing the semen in the uterus close to the time of ovulation. Using frozen semen has many potential advantages: there would be greater choice of stallions, the supply of semen is guaranteed, and the amount of antibiotics used would be reduced compared to fresh semen. However, not all ejaculates can be frozen successfully. This review looks at some of the factors that might affect the successful cryopreservation of semen, including stallion nutrition, the frequency of semen collection, the medium that is added to protect the sperm during freezing, the speed of cooling, etc. It would be helpful to identify ejaculates that will freeze well, but as yet, there are no means of identifying them other than carrying out a test freeze. Artificial insemination (AI) is used frequently in the breeding of sport horses, apart from Thoroughbreds. Most AIs are carried out with cooled semen rather than frozen semen because of the difficulties in identifying a protocol that is suitable for freezing most ejaculates and the necessity to inseminate close to ovulation because of the short life of the thawed spermatozoa. More widespread use of frozen semen would improve biosecurity, allow greater choice of stallions, and offer more flexibility when managing deliveries of semen to the stud. It would even decrease the amount of antibiotics used in semen extenders, since the volume of frozen semen is smaller than when cooled semen is inseminated. However, there is considerable variability in the cryosurvival of spermatozoa from different stallions, leading to the classification of stallions as good or bad freezers. Improvements could be made at the level of stallion nutrition, the semen collection regimen, the extender, the removal of seminal plasma, and the cooling protocol, among others. Stallion sperm membranes are highly susceptible to lipid peroxidation, but research on antioxidants has failed to identify an additive that would benefit all stallions. In the future, biomarkers for sperm freezability could be used as an aid in identifying suitable ejaculates for cryopreservation. [ABSTRACT FROM AUTHOR]

Summary: Simple dilution of semen is the oldest method to process semen for artificial insemination with either fresh or cooled semen. This was developed prior to the optimisation of centrifugation techniques for semen processing through a combination of trial and error and research. Simple dilution is a faster method to process semen, is easy to use and works in many circumstances. For on‐farm semen use a 1:1 (v/v) semen to extender ratio is used. A variety of other techniques have been developed, such as cushion centrifugation, density gradient centrifugation and sperm filtration. Cushion centrifugation is used to concentrate the sperm and allows higher centrifugation speeds than centrifugation alone. This allows for improved recovery rates of sperm. Density gradient centrifugation has been used to select for morphologically normal sperm but suffers from low recovery rates. Processing sperm with this technique can result in improved motility and improved DNA integrity of sperm. Sperm filtration is another method to concentrate sperm without the use of centrifugation; however, it has lower recovery rates compared to cushion centrifugation. This review will discuss these techniques in the application of processing equine semen for either fresh or cooled‐shipped use. [ABSTRACT FROM AUTHOR]

Evaluation of acrosome function in stallion sperm is mostly based on the use of inducers of acrosomal exocytosis (AE), such as the calcium ionophore A23187 or progesterone. Recently, it has been reported that incubation of stallion sperm under presumed capacitating conditions (i.e., medium formulated with calcium, bicarbonate, and bovine serum albumin) using a lactate-only containing medium (Lac-MW) results in a high rate of spontaneous AE in viable sperm (AE/Viable). In the current study, we developed an alternative assay of acrosome function for stallion sperm following the incubation of sperm in a medium formulated only with lactate as an energy substrate (Lac-MW). In Experiment 1, freshly ejaculated stallion sperm was incubated with 10 μM A23187, Lac-MW, or Control, for up to 6 h under capacitating conditions. The percentages of motile sperm, viable sperm, total AE (Total AE), and AE in viable sperm (AE/Viable) were compared among treatment groups. Incubation in Lac-MW, but not with Control or A23187, resulted in a time-dependent increase in the percentage of AE/Viable, as determined by flow cytometry, particularly at 4 and 6 h of incubation (P < 0.05). In Experiment 2, freshly ejaculated sperm was incubated in Lac-MW for up to 6 h, and the occurrence of protein tyrosine phosphorylation and AE/Viable were determined. At 4h and 6h of incubation in Lac-MW, ∼40% of the sperm displayed a protein tyrosine phosphorylation immunofluorescence pattern that coincides with that recently associated with stallion sperm capacitation (i.e., immunofluorescence signal at the acrosome and midpiece). In Experiment 3, the rate of AE/Viable sperm was compared among freshly ejaculated, cool-stored, and frozen/thawed stallion sperm. Except at 2h incubation in Lac-MW, differences in mean AE/Viable among fresh, cool-stored, and frozen/thawed sperm were not observed (P > 0.05). In Experiment 4, the relationship between Total AE (A23187), or AE/Viable (Lac-MW), and in vivo fertility of 5 stallions was determined. A linear relationship was observed between mean AE/Viable and the per-cycle (r = 0.93; P < 0.05) and seasonal (r = 0.66; P < 0.05) pregnancy rates of five stallions used for artificial insemination with cool-stored semen. In Experiment 5, frozen/thawed sperm from subfertile Thoroughbred (TB) stallions, known to carry the susceptibility genotype for Impaired Acrosomal Exocytosis (IAE; FKBP6 A/A-A/A) was evaluated following incubation in Lac-MW. Sperm from subfertile TB stallions with IAE had lower mean AE/Viable, at both 4h and 6h incubation in Lac-MW, when compared to that of fertile control stallions (P < 0.05). Overall, the Lac-MW model validated in the current study may be a useful complementary assay to evaluate the ability of stallion sperm to physiologically undergo AE and to study stallion fertility potential. This acrosome function assay can be used to evaluate fresh, cool-stored, or frozen/thawed stallion sperm, and describes a strong linear relationship with in vivo -fertility of stallions used in artificial insemination programs. • An alternative assay for acrosome function analysis in stallion sperm was validated. • Incubation of stallion sperm in a modified Whitten's medium formulated only with lactate induced both AE/Viable and protein tyrosine phosphorylation. • Cooled storage or freezing/thawing did not negatively affect the occurrence of AE/Viable in stallion sperm. • A linear relationship between AE/Viable and per-cycle and seasonal pregnancy rates of stallions was observed. • The validated assay was used to detect acrosome dysfunction in sperm from highly subfertile stallions. [ABSTRACT FROM AUTHOR]

Simple Summary: Stallion breeding soundness is indicated for infertility or to evaluate the potential to mate or to produce and ship doses of fresh, refrigerated, or frozen semen. This review describes the methods used to evaluate equine semen and their relation to semen production pathways and fertility. Clinical examination is useful for detecting genital tract pathologies, but also for predicting the expected spermatozoa production. Basic semen evaluation in the laboratory includes analysis of the volume, spermatozoa concentration, motility, and morphology. These factors can be used to evaluate the spermatozoa production and its quality, and predict the efficiency of natural mating and the potential fresh or refrigerated semen dose production. The post-thaw quality of some stallions is below standard, despite good fresh semen quality, requiring methods for predicting this outcome. To overcome this concern and the unresolved infertility cases, biotechnologies offer new opportunities. The potential toxicity of seminal plasma can be investigated, and damaged parts of the spermatozoa can be imaged with specific staining and fluorescent microscopy or flow cytometry. These data will deepen the understanding of stallion infertility and enhance basic semen evaluation. Stallion sperm analysis is indicated for infertility diagnosis, pre-sale expertise, production of fresh or frozen doses, and frozen straw quality control. Various collection methods are described, and numerous assays can be performed on semen. Determining an approach for each of these cases is challenging. This review aims to discuss how to obtain relevant clinical results, answering stallion owners' concerns. Semen can be collected with an artificial vagina on a phantom or a mare, by electro-ejaculation under anesthesia, or after pharmacological induction. The collection method influences the semen volume and concentration, while the total sperm number depends on the testicular production and collection frequency. In the seminal plasma, acidity, pro-oxidant activity, and some enzymes have repercussions for the semen quality and its conservation. Moreover, non-sperm cells of seminal plasma may impact semen conservation. Motility analysis remains a core parameter, as it is associated with fresh or frozen dose fertility. Computer-assisted motility analyzers have improved repeatability, but the reproducibility between laboratories depends on the settings that are used. Morphology analysis showing spermatozoa defects is useful to understand production and maturation abnormalities. Staining of the spermatozoa is used to evaluate viability, but recent advances in flow cytometry and in fluorochromes enable an evaluation of multiple intracellular parameters. Spermatozoa protein expression already has clinical applications, for example, as a fertility and freezing ability predictor. At present, stallion semen analysis ranges from macroscopic evaluation to assessing spermatozoa proteins. However, clinically, all these data may not be relevant, and the lack of standardization may complicate their interpretation. [ABSTRACT FROM AUTHOR]

Since the stallion acrosome is very small compared to other species and cannot be properly assessed without additional staining, several labelling techniques were developed to facilitate its assessment. The aim of this study was to compare the Spermac stain (Minitüb GmbH) and a PNA/PSA/PI triple‐staining detected by flow cytometry with regard to method agreement for detecting non‐intact acrosomes within two different extenders. For this purpose, eighteen stallion ejaculates were split in half and diluted with the semen extenders EquiPlus or Gent (Minitüb GmbH) to a final concentration of 50 × 106 sperm/mL, respectively. Subsequently, 126 semen samples were stained with both methods between 4 and 240 h (mean: 63.8 ± 48.9 h) after semen collection. Calculated Intraclass correlation coefficients revealed excellent correlations between both methods for EquiPlus (r =.77, p <.001) and fair correlations for Gent (r =.49, p <.001). Interestingly, flow cytometry detected more non‐intact acrosomes in EquiPlus than in Gent (p <.001), whereas the Spermac stain showed no differences (p =.902) between extenders. The poorer method agreement in Gent could be caused by egg yolk artefacts, which made interpretation difficult, so flow cytometry might be preferred. The differences in detected non‐intact acrosomes between extenders highlighted the importance of establishing adapted laboratory protocols for different extender types in order to generate comparable results. [ABSTRACT FROM AUTHOR]

Glass wool column filtration (GWCF) selects human, bull, boar, dog and buffalo spermatozoa, but reports in the horse are scarce. Single‐layer colloid centrifugation with Androcoll‐E™ is currently the standard procedure to select good‐quality equine sperm. This study was designed to assess GWCF (50 and 75 mg columns; GWCF‐50 and GWCF‐75, respectively) efficacy to select good‐quality sperm from fresh and frozen–thawed equine semen, and to compare its performance with Androcoll‐E™ colloid centrifugation. Percentage total motile (TM), progressively motile (PM), morphologically normal (MN), osmotically competent (HOS+) and acrosome‐intact/osmotically competent (AI/HOS+) sperm were determined. In studies done with fresh semen samples (n = 17), suspensions subjected to GWCF‐50 showed an improvement (p <.05) in PM and HOS+ sperm after selection. With GWCF‐75, an increase (p <.05) in PM, MN and HOS+ sperm was observed. Results with GWCF were comparable or better than with Androcoll‐E™ selection. Sperm recovery was similar between procedures for all semen parameters. Total sperm count recovery was lower after GWCF‐75 (GWCF‐50 = 60.0; GWCF‐75 = 51.0; Androcoll‐E™ = 76.0 million sperm; median; p =.013), but results on total progressive sperm count were similar (GWCF‐50 = 23.0; GWCF‐75 = 27.0; Androcoll‐E™ = 24.0 million sperm; median; p =.3850). Using frozen–thawed semen samples (n = 16), an improvement (p <.05) in TM, PM, NM, HOS+ and AI/HOS+ sperm was observed in GWCF‐75 filtrates. Results were comparable to Androcoll‐E™ centrifugation, except HOS+ that increased (p <.05) only after GWCF‐75. Recovery was comparable for all parameters in frozen samples. GWCF is a simple and low‐cost procedure that selects equine sperm with a quality comparable to colloid centrifugation with Androcoll‐E™. [ABSTRACT FROM AUTHOR]

Summary: Spermiostasis (or sperm accumulation) is an ejaculatory dysfunction of common occurrence in referral reproductive practice with stallions. The accumulation of sperm in the epididymides, ducti deferens, and ampullae results in ejaculates with a high concentration of degenerated sperm. It is estimated that 5% of light‐breed stallions present this condition; however, the incidence is unknown in ponies as this condition has not been reported in these breeds. The present report describes the diagnosis and management of spermiostasis in a 16‐year‐old Shetland stallion presented for semen collection, evaluation, and cooling test. Semen collection resulted in 73 billion total sperm with necrozoospermia and 67% detached heads. Ten serial semen collections gradually improved sperm motility and morphology. There was no evidence of ampullary plugs in the semen filter throughout the collections. An additional semen collection was procured 48 h after collection 10 for a cooling test. Sodium caseinate cholesterol‐loaded cyclodextrin extender resulted in superior sperm motility compared to the native phosphocaseinate extender up to 48 h post‐cooling in two semen containers. Sixty days after the initial visit, the stallion returned for a semen freezing test. The ejaculate was normozoospermic, and the owner reported that the stallion had satisfactory fertility for live cover after discharge from the hospital. The thermal longevity test of the frozen‐thawed resulted in excellent motility after 120 min of storage at 37°C. In conclusion, herein, we described, apparently for the first time, the occurrence of spermiostasis in a pony stallion. We foresee that other practitioners managing similar clinical cases could have comparable outcomes. [ABSTRACT FROM AUTHOR]

Cryopreservation is very important technique in AI centers of stallions, it preserves sperms for long period and spread the superior genetic merits between different animal's breeds. However, using of cryopreserved sperms lead to decreasing the fertility between animals, due to lethal damage of sperms during preservation process; so, this study aimed to use the two different freezing media with decreasing post thawing sperm damage. A total of 54 ejaculates were collected from nine pure fertile Egyptian Stallions (6 ejaculates per stallion), individually housed at a Veterinary Clinic in Giza Government. Semen sample was collected by Missouri AV on a regular basis (two collections / week) during the 2021 breeding season in presence of teaser mare. The collected ejaculates were sent to the laboratory immediately for evaluation by CASA (total concentration, progressive motility, static motility, and sperm abnormalities). Ejaculates were filtrated for removal gel fraction; filtrated ejaculates were diluted by EDTA-glucose media for centrifugation and the resulting sperm pellets were split into 2 equal aliquots and then extended in freezing media. INRA96 Milk-based Extender with glycerol and Egg yolk- based Extender with DMF (dimethylformamide) were used in this study as freezing extenders. Diluted semen was packaged into 0.5 ml straw then cooling at 4 °C and freezing by vapor of liquid nitrogen and after that preserved by freezing in liquid nitrogen container at -196°C. After keeping the frozen straws in liquid nitrogen for one week, at least 2 straws were taken for thawing and evaluating post thawing-freezing motility. Finally thawed-frozen semen was inseminated inside fertile mares for calculation the conception rate after one month. Post thawing motility were evaluated in extended semen by two different extenders. The obtained results showed a change in the motility by decreasing in INRA-diluted semen compared to DMF-diluted semen. Conception rate was recorded after insemination and showed a high significant in DMF-diluted semen than INRA diluted semen. We concluded that the frozen semen with DMF based diluent did not decrease the motility of sperms after thawing and achieved high conception rate when compared with INRA based glycerol diluent. [ABSTRACT FROM AUTHOR]

The use of cryopreserved semen has become a standard technique in equine reproduction and is significantly affected by the stallion’s individual aptitude for the technique. The aim of the study was to compare the effect of different semen handling protocols before cryopreservation on post-thaw semen quality of stallions (n=9) either classified as good (n=5) or poor freezers (n=4). Ejaculates (n=6) were collected, splitted and subjected to five different protocols: Group 1 - ejaculates frozen immediately after collection and centrifugation at room temperature (25°C); Group 2 - ejaculates frozen after cooling at 5°C for 6 hours with centrifugation before storage; Group 3 - ejaculates frozen after cooling at 5°C for 6 hours (slow cooling: 0.1°C/min) without centrifugation before storage; Group 4 - ejaculates frozen after cooling at 5°C for 24 hours with centrifugation before storage ; Group 5 - ejaculates frozen after cooling at 5°C for 24 hours (slow cooling: 0.1°C/min) without centrifugation before storage. Spermatological examination (directly before freezing (T1) and after thawing (T2) included assessment of motility, membrane integrity, acrosomal status and DNA fragmentation index (DFI). Differences between good and bad freezers were observed in all groups. While good freezers had the highest post-thaw progressive motility (T2PM) in Group 1 and 2, poor freezers showed the best results for T2PM in Group 3. Cooled storage for 24 hours resulted in lower T2PM for all groups. Post-thaw DFI and membrane integrity were not affected by treatment group but freezability of the individual stallion. More acrosomal defects were observed in poor freezers as compared to good freezers and after cooled storage for 24h in poor freezers in comparison to cooled storage for 6 hours. In conclusion, good freezers showed the best post-thaw semen quality after immediate centrifugation and cryopreservation or after centrifugation, followed by cooled storage for 6 hours. Post-thaw semen quality of poor freezers benefitted from slow cooling and cooled storage (6 hours) without previous centrifugation. [ABSTRACT FROM AUTHOR]

Equine semen cryopreservation is one of the major procedures for the genetic conservation of rare and endangered genotypes. The current study was conducted to evaluate the effect of egg yolk plasma (EYP) enriched with β‐carotene as an antioxidant supplement on INRA‐96 extender regarding freezing Arabic stallion sperm. For this purpose, β‐carotene various concentrations were utilized as a supplementary ingredient in formulating the diets of laying hens. Birds were randomly divided into four groups, fed with 0 (control), 500, 1000 and 2000 mg/kg in a supplemented diet with β‐carotene. Subsequently, various variants of enriched extender (INRA‐96 + 2.5% glycerol [G]) were gained by adding 2% EYP from four treatment groups. The sperm characteristics, including motility, viability, morphology, plasma membrane integrity (HOS test), lipid peroxidation (MDA) and DNA fragmentation, were evaluated after thawing. According to the results obtained in this study, the addition of EYP from T2 and T4 (500 and 2000 mg/kg of β‐carotene in hens' diet) to the extender (INRA‐96 + 2.5% G) leads to an increase in total motility (50.50% and 49.49%, respectively), progressive motility (32.6% and 31.8%, respectively), viability (68.7% and 66.1%, respectively) and plasma membrane integrity (57.7% and 50.6%, respectively). Moreover, lipid peroxidation (1.3 and 1.4 nmol/mL, respectively) and DNA fragmentation (8.6% and 9.9%, respectively) were diminished using the mentioned treatments. However, sperm morphology was not affected by the treatments. In the current study, we concluded that the optimal concentration of β‐carotene in the laying hen's diet (500 mg/kg) could reveal the best results about sperm quality. So, EYP enriched with β‐carotene acts as a valuable natural and safe supplementary material that could be exploited for enhancing stallion sperm quality in cryopreservation conditions. [ABSTRACT FROM AUTHOR]

The use of microfluidic technology is increasing in artificial reproduction technologies: With a small amount of semen, it allows for the selection of sperm with the best characteristics of kinetics, morphology and chromatin integrity. The ZyMot Multi (850 μl) is the most popular device of ZyMot Fertility Inc. To date, it was proven to be a valid instrument for sperm selection for in vitro fertilization and intracytoplasmic sperm injection in men. The aim of this study was to test the efficacy of the ZyMot Multi (850 μl) for stallion semen. Frozen–thawed semen from 15 stallions that were previously classified as being of 'good fertility' (GF; n = 8; pregnancy rate ≥ 40%) and 'poor fertility' (PF; n = 7; pregnancy rate < 20%), respectively, was used. Each ejaculate was assessed before and after microfluid recovery for kinetics (CASA), membrane integrity (MI) (SYBR14/PI), membrane functionality (MF) (HOS test), acrosome integrity (Spermac Stain Kit), morphology (Spermac stain kit), mitochondrial membrane potential (MMP) (JC‐1) and chromatin integrity (aniline blue staining). Sperm concentration was reduced after sperm recovery in both groups, but more markedly in frozen–thawed semen of PF stallions (p <.05). Microfluid recovery increased total motility, MI, MF and MMP. While there was a significant increase in the percentage of progressively motile sperm after sperm microfluid recovery, there was a decrease in DAP, DSL, VAP, VSL, LIN, WOB and ALH (p <.05). A slight increase (p <.05) was detected in beat‐cross frequency. The present results suggest that the ZyMot Multi (850 μl) device selects a specific sperm population from any stallion ejaculate with motile sperm and could therefore be a valid tool for in vitro testing with the aim to predict the fertility of frozen–thawed stallion semen. [ABSTRACT FROM AUTHOR]

Simple Summary: To determine the semen quality after cooling and freezing, the first and second ejaculates of the season were collected 1 h apart. After collection, the gel-free semen volume, concentration, total number of sperm, and sperm morphology were determined. An aliquot of the ejaculate was extended and cooled for 48 h; a second aliquot was processed, centrifuged, and then cooled for 48 h; and a third aliquot was processed and then frozen. The sperm parameters were assessed pre-(0 h), 24 h, and 48 h post-cooling and before and after freezing. The first and second ejaculates of the season collected 1 h apart varied in quantity but not in quality. Centrifugation prevented a reduction in the sperm quality after cooling. The semen freezing did not vary between the first and second ejaculates. This study aimed to assess the semen quality after the cooling and freezing of the first and second ejaculates of the season, which were collected 1 h apart. After collection (n = 40 ejaculates), the gel-free semen volume, concentration, total number of sperm, and sperm morphology were determined. An aliquot of each ejaculate was extended and cooled for 48 h; a second aliquot was cushion-centrifuged and cooled for 48 h; and a third aliquot was processed and then frozen. The total motility (TM) and progressive motility (PM), plasma membrane integrity (PMI), and high mitochondrial membrane potential (HMMP) were assessed pre-(0 h), 24 h, and 48 h post-cooling and before and after freezing. The second ejaculate had a lower gel-free semen volume (p = 0.026). The sperm concentration was greater in the first than in the second ejaculate (p < 0.001). The sperm morphology was similar between the ejaculates (p > 0.05). Cushion-centrifugation prevented a reduction in the TM, PM, and PMI over time (p < 0.05). The TM, PM, and PMI decreased after freezing but not between the ejaculates (p > 0.05). The first and second ejaculates of the season, which were collected 1 h apart, varied in quantity but not in quality after cooling and freezing. [ABSTRACT FROM AUTHOR]

Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation‐produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been debated by researchers over the last decade. The objective of this study was to evaluate the relationship between various sperm morphologies and ROS production in fresh and cooled stallion semen by employing the novel method of imaging flow cytometry for stallion semen assessment. For evaluation of fresh semen, single ejaculates (n = 5) were collected from four resident stallions at the University of California, Davis. For the evaluation of 24‐h cool‐stored semen, single ejaculates were collected from stallions at Texas A&M University (n = 5) and shipped to the University of California, Davis overnight for evaluation. Ejaculate volume, sperm concentration and motility parameters were recorded. Samples were co‐stained for viability and ROS detection with SytoxGreen™ and dihydroethidium (DHE), respectively, and evaluated with the Amnis® ImageStream® system (Luminex Corporation). Antimycin, an electron transport chain inhibitor that triggers ROS production (1 μM), was used as a positive control for DHE, while dead cells (2× snap frozen in liquid nitrogen) served as a positive control for SytoxGreen™. Unstained samples were also evaluated as controls. Imaging flow cytometric analysis was performed with the ideas® software (Luminex Corporation). Evaluated morphologies included abnormal head (AH), abnormal midpiece (AM), abnormal tail (AT), proximal cytoplasmic droplet (PD), or distal cytoplasmic droplet (DD), and morphologically normal (MN) cells. For fresh semen, an additional abnormality, coiled tail and midpiece (CTM) was assessed; 24‐h cool‐stored semen did not contain enough viable CTM cells for analysis. Only cells with obvious, single abnormalities were selected for the first portion of analysis to minimize subjectivity. Mixed effects modelling was used to evaluate the relationship between each morphologic classification and the corresponding DHE fluorescence intensity. Compared to the MN population, ROS production was significantly higher in viable cells with AH, PD and AM (p <.0001) in both fresh and cooled semen. CTM cells had significantly higher levels of ROS production compared to MN cells in fresh semen (p <.0001). There was no significant difference in ROS levels between MN cells and AT and DD cells in either fresh or cooled semen (p >.05). These results suggest that ROS generation is indicative of abnormal cell morphology and function and confirm that imaging flow cytometry is a valuable tool for the assessment of stallion semen. [ABSTRACT FROM AUTHOR]

Objective: The semen quality of stallions including sperm motility is an important target of selection as it has a high level of individual variability. However, effects of the molecular architecture of the genome on the mechanisms of sperm formation and their preservation after thawing have been poorly investigated. Here, we conducted a genome-wide association study (GWAS) for the sperm motility of cryopreserved semen in stallions of various breeds. Methods: Semen samples were collected from the stallions of 23 horse breeds. The following semen characteristics were examined: progressive motility (PM), progressive motility after freezing (FPM), and the difference between PM and FPM. The respective DNA samples from these stallions were genotyped using Axiom Equine Genotyping Array. Results: We performed a GWAS search for single nucleotide polymorphism (SNP) markers and potential genes related to motility properties of frozen-thawed semen in the stallions of various breeds. As a result of the GWAS analysis, two SNP markers, rs1141327473 and rs1149048772, were identified that were associated with preservation of the frozen-thawed stallion sperm motility, the relevant putative candidate genes being NME/NM23 family member 8 (NME8), olfactory receptor family 2 subfamily AP member 1 (OR2AP1), and olfactory receptor family 6 subfamily C member 4 (OR6C4). Potential implications of effects of these genes on sperm motility are herein discussed. Conclusion: The GWAS results enabled us to localize novel SNPs and candidate genes for sperm motility in stallions. Implications of the study for horse breeding and genetics are a better understanding of genomic regions and candidate genes underlying stallion sperm quality, and improvement in horse reproduction and breeding techniques. The identified markers and genes for sperm cryotolerance and the respective genomic regions are promising candidates for further studying the biological processes in the formation and function of the stallion reproductive system. [ABSTRACT FROM AUTHOR]

Simple Summary: ProAKAP4 is the precursor of AKAP4 protein, whose principal function is to anchor protein complexes to regulate the long-term motion and flagellum structure of mammal spermatozoa. The objective of the present study was to evaluate how proAKAP4 sperm concentrations correlate with the motion parameters of equine spermatozoa. We showed that proAKAP4 concentrations are associated with motility and velocity parameters. Low proAKAP4 concentrations reflect a loss of spermatozoa functionality. In conclusion, proAKAP4 concentrations are associated with better motion parameters in stallions. ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyzed. Spermatic motility descriptors were obtained by the CASA system, and four motile subpopulations (SP) with specific motility patterns were statistically identified. ProAKAP4 concentrations were evaluated by ELISA. The relationship between motility descriptors of sperm subpopulations and proAKAP4 concentrations was evaluated. Following a hierarchical cluster statistical analysis, ejaculates were divided into two groups according to their proAKAP4 concentrations, either having low proAKAP4 concentrations (5.06–35.61 ng/10M spz; n = 23) or high (39.92–82.23 ng/10M spz; n = 25) proAKAP4 concentrations (p < 0.001). ProAKAP4 concentrations were positively correlated (p < 0.05) with total and progressive motility, as well as with parameters of velocity. ProAKAP4 amount also showed a negative correlation (p < 0.05) with sperm motile subpopulation number 3, which was the subpopulation with the lowest velocity parameters. In conclusion, proAKAP4 concentration in stallion semen positively reflects sperm progressive motility with the functional velocity kinematic descriptors. Concentrations of proAKAP4 higher than 37.77 ng/10M spz were correlated with a very good quality frozen/thawed stallion semen. [ABSTRACT FROM AUTHOR]

Background: Artificial insemination with chilled sperm is a reproductive biotechnology often used in horse breeding. However, maintaining the acceptable sperm quality after cooling, which affects the frequency of pregnancy, remains an urgent problem at the present time. Therefore, clarifying the role of important physiological metabolites, such as L-carnitine and nitric oxide, is a significant aspect in terms of possible improvement of the quality of cryopreserved sperm. Methods: The study included 35 breeding stallions, 27 of which were stallions of the Arabian breed and 8 were of the Soviet heavy draft breed. The content of total, free and bound carnitine and NO metabolites was determined by photometric method in the samples of seminal plasma and blood plasma. The correlation coefficient between the content of L-carnitine and the level of NO metabolites was also determined. Result: We have found a direct moderate correlation between the level of total and free L-carnitine in the seminal plasma and blood plasma. The study has also revealed a positive correlation between the level of all L-carnitine fractions and NO metabolites in seminal plasma. In addition, there was a positive correlation between the total L-carnitine of blood plasma and NO metabolites in seminal plasma. [ABSTRACT FROM AUTHOR]

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. • Membrane stabilizers were evaluated in stallion semen. • CLC improved post-thaw sperm quality parameters. • Results encourage potential use in reproductive biotechnologies. [ABSTRACT FROM AUTHOR]

In vitro embryo production (IVEP) via Ovum Pick-Up (OPU) and Intracytoplasmic Sperm Injection (ICSI) has become a popular breeding technique in Warmblood mares because of the high success rate and several practical advantages. IVEP offers a solution for a variety of reproductive issues including, but not limited to, sub-fertility in stallions or mares, poor quality or scarce frozen semen, difficulty in synchronizing donor and recipient mares, and inefficient use of recipient mares. In 515 OPU-ICSI sessions performed in our facility in 2021, a mean of 25.9 antral follicles were aspirated yielding an average 13.8 immature oocytes, which were shipped overnight to a specialized ICSI laboratory (Avantea). One or more blastocysts (range: 0–13 blastocysts) were produced from 78% of procedures with a mean of 2.12 blastocysts per session; the likelihood of pregnancy after transfer of a cryopreserved thawed IVP blastocysts in 2021 (n = 781) was 77.7%. Several donor mare, recipient mare, stallion and embryonic factors influence the likelihood of producing an in vitro blastocyst or achieving pregnancy. Approximately 60% of the transferred IVP blastocysts yield a foal; moreover, neither gestation length nor the health of foals is noticeably influenced by IVEP. On the other hand, a skewed sex ratio towards colts is apparent among IVEP foals resulting from day 7 but not day 8 embryos, suggesting that male embryos develop more rapidly in vitro. Although serious complications after OPU are uncommon, owners should be aware of their existence, because some complications can be life-threating. • In vitro embryo production in mares is successful in 78% of OPU-ICSI procedures. • The pregnancy rate after transfer of a cryopreserved IVP equine embryo is above 70%. • Likelihood of IVEP and pregnancy is influenced by several donor mare, recipient mare, stallion and embryonic factors. • IVEP appears to be sex dependent in our system. [ABSTRACT FROM AUTHOR]

Adding antioxidants to semen extender has become one of the major strategies to reduce the damage caused by freezing to stallion sperm. Isoespintanol (Iso) is a natural antioxidant that is known as a good free radical scavenger. The aim of this research was to evaluate the effect of supplementation of freezing media with Iso on functional integrity of equine semen. Semen from five Colombian Creole horses was frozen with 0, 10, 20, 30, 40 and 50 µM Iso. Post-thawing motility and kinetics, structural membrane integrity (SMI), normal morphology (NM) and functional membrane integrity (FMI) of sperm were evaluated. Seminal quality (SQi) and seminal velocity (SVi) indexes were calculated. Through a flow cytometric analysis, mitochondrial membrane potential (ΔΨM), viability and DNA integrity of frozen-thawed semen were assessed. General linear models were adjusted, and means comparisons were performed using Tukey's test. The significance level was P<0.05. No differences for post-thaw sperm quality parameters were found, except for total motility, which showed a reduction by adding 40 µM Iso (P<0.05). However, the estimation of indexes, showed that supplementation with 20 or 30 µM Iso before semen freezing, can increase post-thaw SQi and SVi values of some horses. A higher proportion of high-∆ΨM sperm for 20 µM Iso was found. It is concluded that Iso can improve the quality and mitochondrial activity of frozen-thawed equine semen, however its effect can vary according to the horse and in high concentrations it could have detrimental effects. [ABSTRACT FROM AUTHOR]

This study aimed to assess the semen ubiquitin levels of stallions with good (GF) and poor semen freezability (PF) and to evaluate the relationship between sperm ubiquitination and sperm morphological defects. Five ejaculates from eight adult stallions (n = 40) were collected and cryopreserved. Then, the ubiquitin level in equine sperm cells was assessed by immunohistochemistry with epifluorescence microscopy, and sperm morphology was assessed by differential interference contrast microscopy. Sperm cells were classified according to the intensity (classification 1: from I to IV; I = very low ubiquitin intensity and IV = very high ubiquitin intensity) and location of ubiquitin staining (classification 2). Statistical analyses were performed using SAS software (version 9.4), and p ≤.05 was considered significant. We observed that PF stallions showed higher percentages (p <.05) of sperm cells with high ubiquitination (11.82% of ubiquitin intensity grade I, 39.13% of ubiquitin intensity grade II, 27.25% of ubiquitin intensity grade III, and 20.67% of grade IV), while GF stallions showed higher percentages (p <.05) of sperm cells with lower staining intensity (28.52% grade I, 59.83% grade II, 7.92% grade III, and 7.02% grade IV). Furthermore, for PF stallions, 23 significant correlations were detected (p <.05) between sperm abnormalities and ubiquitin intensity in different sperm regions. Increased ubiquitination of the sperm head, midpiece, and tail was positively correlated with their respective morphological defects. We concluded that high sperm ubiquitin levels are observed in ejaculates from stallions with poor semen quality (poor freezability), and ubiquitin marking in specific cellular locations can identify sperm morphological defects. [ABSTRACT FROM AUTHOR]

The use of frozen semen lowers the risk of disease transmission, eliminates geographical limitations and supports the implementation of genetic resource protection programs. However, due to the very rare use of frozen semen from Hutsul stallions, their genetic material is not secured in sperm banks, and very little information is available about their semen, including its suitability for cryopreservation, and sperm survival rates after thawing. The aim of this study was to analyse basic parameters such as sperm motility, vitality and morphology in diluted-stored and post-thawed Hutsul semen, using a CASA system. There were no differences in sperm motility (P = 0.3372) or morphology between the groups, although the progressive motility was higher in thawed semen (P = 0.0151), while the sperm vitality was higher in diluted-stored semen (P = 0.00517). This study demonstrates that semen from Hutsul horses is suitable for cryopreservation, thus supporting the creation of a sperm bank as a genetic reserve for representatives of this breed. [ABSTRACT FROM AUTHOR]

Anti-Müllerian Hormone (AMH) has been linked to reproductive tract abnormalities in mares and stallions. This study aimed at evaluating AMH as a biomarker for two reproductive conditions in mares. In the first part of this study, plasma AMH was evaluated as an early indicator of the onset of cyclicity in mares in the transitional period from the anovulatory phase during winter anoestrus to the cyclic phase during the breeding season. Ten mares between 8 and 17 years old were included in the experiment which lasted from mid-February until the end of April. Ovarian activity was monitored with ultrasonography three times per week, the detection of a corpus haemorrhagicum/luteum was documented and antral follicle counts (AFC) were recorded. Blood samples were collected weekly by jugular venipuncture during the whole study period to compare AMH concentrations before and after the first ovulation of the year. The second objective was to evaluate if plasma AMH concentrations in middle-aged mares are linked to fertility and could serve as a prognostic marker in that age group. A total of 41 privately-owned clinically sound mares aged between 12 and 21 years of various breeds were inseminated with fresh or frozen semen. Mares were scanned between day 14 and 20 and the "early pregnancy rate" included only positive pregnancy examinations after the first observed cycle in the season of each mare. Potential associations between the early pregnancy rate in the first cycle and the explanatory factors AMH concentrations, age, status of the mare, stud, development of post-breeding endometritis, number of inseminations and semen type were analysed using logistic regression models. In the first part of the study, correlation between AMH and AFC for the whole study period (P = 0.0002, ρ = 0.55) as well as prior to (P = 0.008, ρ = 0.58) and after the first ovulation (P = 0.0007, ρ = 0.69) were observed. However, AMH concentrations before and after the first ovulation of the year were not statistically different. The second part of the study revealed no association between early pregnancy rate and AMH concentrations or any of the other mentioned factors. In conclusion, this study showed no evidence of a difference between AMH concentrations before and after the first ovulation of the year thus not supporting the use of AMH as a biomarker to predict the onset of cyclicity in mares. We could furthermore not show a relationship between plasma AMH concentrations and early pregnancy rates in this cohort of animals. • Our data did not demonstrate that AMH could predict the onset of cyclicity in mares a highly significant correlation between AMH and antral follicle counts (AFC) was observed. • We found no difference between AMH concentrations before and after the first ovulation. • We found no relationship between AMH concentrations and early pregnancy rates in mares over 12 years old. [ABSTRACT FROM AUTHOR]

This work aimed to study the influences of different extender types on the quality and fertilizing capacity of stallion semen to determine the best extender which may enhance the fertilization capacity of stallion semen. Three healthy stallions aged 4-10 years and sixty mares aged 3-12 years were selected after the breeding soundness examination to accomplish this study. All the animals were with a good general health condition without any history of reproductive failures or disturbances. The external and internal genitalia were examined using the rectal palpation and ultrasonography to avoid any disturbances in the reproductive tract that might affect mare’s fertility. Semen samples were collected using artificial vagina and was evaluated, then diluted by four types of extenders (botusemen, botusemen special, botusemen gold and botusemen turbo ) and used for mares insemination to determine the influence of extender on quality of semen and conception rate.This study concluded that, the extenders types can influence the fertilizing capacity in mares through its effect on active forward motility of the sperm, dead and live sperm % and the conception rate. The extenders that contain skimmed milk could increase the fertility and conception rates of stallion semen. [ABSTRACT FROM AUTHOR]

Different stallions exhibit a high level of variation in the ability of their sperm to survive cryopreservation. A large fraction of stallions show poor post-thaw sperm motility, and their semen is not suitable for commercial freezing. In this study, we hypothesized that the presence of sperm-bound antisperm antibodies (ASAs) was associated with poor cryosurvival of stallion sperm. Our objective was to assess the level of ASA binding to stallion sperm, and determine if it was associated with good or poor sperm cryosurvival. In Experiment 1, cooled shipped semen from 27 stallions was frozen using three commercial semen extenders. Sperm motility, membrane integrity, acrosome integrity and apoptosis were evaluated before and after freezing for each aliquot. In addition, the percentage of ASA-bound sperm was evaluated post-thaw. In Experiment 2, semen from 22 stallions was frozen immediately after collection a single formulation of semen extender. Sperm motility and ASA binding were evaluated post-thaw. The results of both experiments showed similar findings. The frequency of ASA-positive samples was higher among stallions with poor sperm cryosurvival (Exp. 1 and 2 = 6/11, 54.5%) than for good sperm cryosurvival (Exp. 1 = 0/16, 0%; Exp. 2 = 1/11, 9.1%). The percentage of IgG- and IgA-bound sperm was also higher in stallions with poor sperm cryosurvival in both experiments (P < 0.05). Post-thaw sperm motility, velocity and distance parameters were lower in ASA-positive than ASA-negative stallions (P < 0.005). No effect of the semen extender used was observed. In addition, stallions with ASAs had a higher percentage of apoptotic sperm than stallions without ASAs. The presence of sperm-bound ASAs was associated with poor cryosurvival for stallion spermatozoa. Thus, it may be beneficial to evaluate stallions for binding of ASAs prior to freezing to offer and indicator of the prognosis for cryosurvival. • 54.5% of stallions with poor sperm cryosurvival had high ASA binding • Less than 10% of stallions with good sperm cryosurvival had ASA binding • Sperm-bound ASAs were associated with poor cryosurvival for stallion sperm • Identifying ASAs binding prior to freezing may offer a prognosis for cryosurvival [ABSTRACT FROM AUTHOR]

Two substances at two concentrations each were added to frozen-thawed stallion epididymal spermatozoa to improve motility indicators, which were assessed 30, 60, 120, 180 and 240 min after thawing. Pentoxifylline was added at concentrations of 3.6 mM (1 mg/ml) and 7.18 mM (2 mg/ml). Both concentrations had a positive effect on total and progressive motility of spermatozoa throughout the study. The other substance, caffeine, was added at concentrations of 2 mM (0.4 mg/ml) and 5.5 mM (1 mg/ml). Both concentrations had a positive effect on total and progressive motility of spermatozoa only 30 and 60 min after thawing. Subsequently, 180 min after thawing, improvement was found only in total sperm motility. [ABSTRACT FROM AUTHOR]

Nutritional supplements are widely used in the equine industry with the aim of improving horse health, sports or reproductive performances. Over the years, a number of studies have focused on investigating the effects of several dietary compounds on the quality and preservation of stallion semen. This paper reviews the literature available on the use of nutritional supplementation for the improvement of reproductive performance and semen quality in equine species, critically appraising the benefits and negative effects of several compounds found in complementary feeds such as PUFAs from different sources, vitamins and antioxidants, carnitine and botanical extracts. Different nutraceuticals have been highlighted to improve stallion fertility by providing optimal levels of antioxidants, with the most promising results obtained by the combination of PUFAs and antioxidants that resulted to be essential for the maintenance of normal reproductive functions and the reduction of cryodamage in cooled and frozen equine semen. [ABSTRACT FROM AUTHOR]

Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfasalazine (SS), a specific inhibitor of SLC7A11 antiporter. Viability, motility, immunolocalization of tyrosine phosphorylated proteins and the ability to bind to heterologous zonae pellucidae were evaluated. Thawed sperm from seven stallions (2 ejaculates/stallion) was washed and resuspended in Tyrodes media; each thawed ejaculate was divided in Control (CTR) and 3 samples supplemented with: 0.5 mM Cystine (CysS), 500 μM Sulfasalazine (SS) and 0.5 mM CysS + 500 μM SS (CysS + SS). After 1 h of incubation at 37 °C, samples were washed twice, resuspended in capacitating BWW medium and incubated at 38 °C under 5% CO 2. After 30 and 60 min, sperm motility, viability and tyrosine phosphorylated protein immunolocalization, used as capacitation status index, were evaluated. After 30 min of capacitation, 4 × 105 sperm were co-incubated with denuded pig oocytes in capacitation medium for 30 min for the heterologous binding assay. None of the sperm parameters studied (motility, viability and tyrosine phosphorylation) showed any difference respective to control. The number of sperm bound per oocyte (mean ± SEM) tended to increase in CysS group (44.0 ± 12.3) respect CTR (40.8 ± 10.8) while decreased in SS group (32.4 ± 7.8) (p < 0.01). Moreover, CysS + SS group showed a lower binding rate (32.0 ± 10.0) compared to CysS (p < 0.001). Our results suggest that CysS supplementation of thawed stallion sperm can influence their ability to bind to heterologous zona pellucidae as the inhibition of CysS incorporation by SLC7A11 reduced the number of sperm bound per oocyte. This effect does not seem to be ascribed to a modification of sperm motility, membrane integrity and tyrosine phosphorylation. • Cystine and/or sulfasalazine did not influence sperm viability and motility in thawed stallion sperm after capacitation. • Cystine and/or sulfasalazine did not affect tyrosine phosphorylation after sperm capacitation. • The inhibition of SCL7A11 antiporter significantly decreased the number of sperm bound to heterologous zonae pellucidae. [ABSTRACT FROM AUTHOR]

Dr. Edward Squires, the recipient of the 2024 IETS Pioneer Award, has made significant contributions to the field of equine reproduction. He conducted research on various aspects of mare and stallion reproduction, including follicular and luteal development, embryo transfer, and the use of progestin altrenogest. Dr. Squires also investigated seasonal changes in hormone levels and the barriers to using frozen semen. He has published numerous articles and chapters in textbooks, and has lectured extensively both nationally and internationally. Dr. Squires has received several awards for his work and continues to be involved in teaching and research. [Extracted from the article]

Background: Seminal plasma (SP) plays a crucial role in sperm protection and functionality. However, the effect of SP on the sperm cryopreservation is dependent on the stallion and SP composition. The use of epididymal spermatozoa incubated in the presence of SP could help the identification of the components of SP that are able to confer protection upon the spermatozoa during freezing. Objective: The aims of this study were (i) to identify SP components involved in the potential protection of epididymal spermatozoa during the freeze‐thawing process and (ii) to identify and evaluate the proteins likely related to sperm freezability, using two‐dimensional difference gel electrophoresis (2D‐DIGE). Materials and Methods: Epididymal spermatozoa from 4 stallions were incubated with SP (80%, v/v) or without SP (control) before freezing. Sperm parameters were evaluated after thawing (viability, chromatin condensation, acrosomal integrity, reactive oxygen species [ROS]) and SP composition: total antioxidant capacity (TAC), fatty acid composition, total protein concentration, and protein components by 2D‐DIGE. Results: After thawing, the proportions of viable and acrosome‐intact spermatozoa were higher than control when SP from two stallions was used (F and O). The SP of all stallions reduced ROS production in comparison with the control. After analyzing the SP components, it was found that total protein concentration, TAC, polyunsaturated fatty acids (PUFA), and eight specific proteins identified by 2D‐DIGE were different between stallions. Discussion: These studies allow the identification of SP components that could be involved in sperm protection or cryotolerance. Use of this information could help in the selection of stallions according to their semen freezing capacity. Conclusion: The composition of the SP probably contributes to semen cryotolerance capacity. Total protein, TAC, PUFA, and some proteins such as cysteine‐rich secreted protein 3 could be used as biomarkers for the selection for sperm cryotolerance. [ABSTRACT FROM AUTHOR]

The present study was conducted to evaluate the functional membrane integrity of cryopreserved spermatozoa from stallions belonging to two contrasting motility groups (high and low motility groups). A dual staining method comprising of carboxy fluoroscein diacetate (CFDA) and propidium iodide (PI) was used for this purpose. Stallions were classified into either high (post thaw progressive motility e"35%) or low (post thaw progressive motility <25%) motility groups. The proportion of membrane intact spermatozoa was significantly (P<0.05) higher in high motility group. Interestingly, it was observed that the proportion of moribund population was significantly (P<0.05) higher in low motility group as compared to high motility group. It was inferred that higher proportion of moribund spermatozoa might be one of the important contributing factors for poor sperm motility in stallions belonging to low motile group. [ABSTRACT FROM AUTHOR]

Six ejaculates from each three adult stallions were collected via artificial vagina using estrus jenny as dummy. Caffeine was added to semen extender at 0.1, 0.5 and 2 mM during pre-freeze stage and cryopreserved by following standard protocol. The sperm kinetic parameters, membrane functionality and integrity were evaluated with computerassisted sperm analysis, a hypo-osmotic swelling test and epifluorescent microscopy, respectively. The present study revealed that, adding caffeine to stallion semen extender at 0.1, 0.5 and 2 mM levels had positive impact on seminal parameters compared to the control semen. Sperm kinetics (VAP, VSL and STR) and motility were increased in the caffeine treated groups in comparison to the control group. Among these, 2mM caffeine supplemented group the post-thaw sperm motility and other functional attributes have significantly improved compared to the other treatment and control groups. [ABSTRACT FROM AUTHOR]

The Thoroughbred and Standardbred studbooks are the largest in New Zealand, where the production of horses is pasture based. Each racing studbook is closed, and both have well structured systems in place for recording breeding data. There are significant pressures on the Thoroughbred and Standardbred breeding industries with declining broodmare numbers, and increasingly large book sizes for popular stallions. The breeding season for Thoroughbreds is very short, with disparity between the official breeding season and the physiologic breeding season for mares. These issues are confounded by variable gestation lengths of mares, making it difficult for mares to maintain yearly foaling patterns. However, the reproductive efficiency of Thoroughbred mares is improving, mainly due to veterinary and stud management practices such as those to ensure that dry mares are cycling at the start of the breeding season, foaling mares are bred on foal heat, and that mares are kept in good body condition at breeding. There is also a bias towards breeding younger mares with high fertility in preference to older mares, unless they have desired genetics or successful offspring. Careful management of popular Thoroughbred stallions ensures that large books of mares can be covered by natural service. In contrast, Standardbred stallions are collected every-other-day using an artificial vagina, allowing the breeding of mares at distant locations by artificial insemination, using chilled or frozen semen. Breeding horses kept at pasture under New Zealand conditions requires excellent stud management and veterinary management to achieve good outcomes. [ABSTRACT FROM AUTHOR]

Present study aimed to investigate the effect of adding antioxidants, cysteine and ascorbic acid on the levels of glutamic oxaloacetic transaminase (GOT), glutamic‐pyruvate (GPT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and γ‐glutamyl transpeptidase (GGT) enzymes of post‐thawed stallion sperm. Ten ejaculates were collected each from four healthy stallions and cryopreserved using HF‐20 freezing extender containing either 0 mg/ml cysteine or ascorbic acid, 0.5 mg/ml cysteine and 0.5 mg/ml ascorbic acid. All samples in freezing extender containing cysteine or ascorbic acid or none of them were assessed for sperm motility, viability, plasma membrane integrity, morphology and enzymes concentration. The ALP, LDH and GGT were significantly higher in 0‐group compared with cysteine and ascorbic acid groups. The sperm motility of frozen‐thawed semen with 0‐group was significantly better compared with cysteine and ascorbic acid groups. The variation on viability, sperm membrane integrity and morphology were insignificant between all treated groups. Therefore, these enzymes were reduced when using antioxidants in the freezing extender. Results of the present study suggest that concentration of ALP, LDH and GGT enzymes could be used as parameters for prediction of frozen‐thawed stallion semen. This manuscript aims to evaluate the effect of cysteine and ascorbic acid to HF‐20 extender on the frozen semen enzyme and using these enzymes as markers of frozen semen quality in stallion's semen. It shows that the level of ALP, LDH and GGT enzyme steadily correlated with post‐thawed semen quality. The samples treated with cysteine and ascorbic acid showed a deleterious effect of sperm enzyme concentration and function compared with control group. [ABSTRACT FROM AUTHOR]

Background: Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Gums have been suggested as an alternative cryoprotectant to glycerol for stallion spermatozoa. Therefore, the present experiment was designed to verify whether the effect of addition of cashew gum (CG), or nanoparticles (NP) containing CG, to the extender before cooling on sperm quality in stallion semen. Ejaculates from 6 stallions were extended and split between six treatment groups (control, a-tocopherol [TOC], CG1, CG0.5, NP1 and NP0.5), stored in cryotubes at 4 °C. Results: Aliquots were analysed by computer-assisted sperm motility analysis on the day of collection, and after 24 h and 48 h of cold storage. After 48 h, the total motility with NP1 (78.53 + 6.31%) was similar to control 85.79 + 6.31% at 0 h. The same pattern was observed for progressive motility. Membrane integrity assessed by flow cytometer was similar between control, TOC and G1 at all storage times. The DNA fragmentation in the control group increased at all time points, whereas chromatin integrity was maintained after 24 h in TOC and NP0.5 compared to 0 h. There was no increase in the proportion of live spermatozoa producing hydrogen peroxide, but there was a tendency for an increased proportion of spermatozoa in the live superoxide category in CG1 after 24 h cooled storage. Conclusions: The addition of CG or CG-derived NP to extender for stallion semen was not harmful to the sperm cells. [ABSTRACT FROM AUTHOR]

In Experiment 1, the effects of glucose concentration in extender (0 mM, 67 mM, 147 mM, 270 mM; G0, G67, G147, and G270, respectively) and storage temperature of extended semen (5, 10, 15 and 20 °C) were evaluated after storage for up to 5 days (T0h to T120h). For all time points tested, mean total (TMOT) and progressive (PMOT) sperm motility were lower in G0 than all other treatment groups (P < 0.05). Mean curvilinear velocity (VCL) was lower in G0 than other treatment groups at all time points tested except T0h (P < 0.05). Mean percentage of plasma membrane/acrosome intact sperm (VAI) was similar among treatments at T0h, T72h, and T120h (P > 0.05). Mean TMOT and PMOT, were lower for semen stored at 20 °C than all lower storage temperatures (P < 0.05) at all time points. In Experiment 2, semen was stored at 10 °C in extender containing no added glucose (G0) or 147 mM glucose (G147). Following storage, semen was centrifuged and resuspended in extender containing no added glucose (G0 – G0 or G147 – G0, respectively) or 147 mM of glucose (G0 – G147 or G147 – G147, respectively). Mean TMOT, PMOT, and VCL were higher in G147 than G0 at all time periods tested (P < 0.05), whereas mean VAI was similar between these treatment groups throughout the experiment (P > 0.05). Mean TMOT and PMOT were higher in G0 – G147 than G0 – G0 at T72h and T120h (P < 0.05) and mean VCL was higher in G0 – G147 than G0 – G0 for all time periods. Mean TMOT, PMOT, and VCL were higher in G147 – G147 than G147 – G0 at all time points tested (P < 0.05), whereas mean VAI was similar between these two treatment groups for each of the time points (P > 0.05). In Experiment 3, the minimum concentration of glucose required to maintain sperm quality following long-term cooled storage (T120 h) was evaluated (G0, G5, G10, G20, G40, G67, G147 mM). At T120 h, mean TMOT was lowest in G0, G5, G10, and G20 (P < 0.05), whereas mean PMOT and VCL were lower in G0, G5, G10, and G20 than in G40, G67, and G147 (P < 0.05). Mean VAI was higher in G10 than G67, but similar among G10 and other treatment groups (P > 0.05). In conclusion, the absence of added glucose in extender reduced the motion characteristics of stallion sperm during long-term storage (5 days), but VAI was not affected. The use of temperatures between 5 and 15 °C for long-term storage (5 days) best maintained sperm motility and VAI. The threshold concentration of added glucose in extender required to optimize sperm motion characteristics was 40 mM. • Glucose concentration in extender affected sperm motility but not viability. • Storage temperatures, 5, 10 or 15 °C, preserved better sperm quality than 20 °C. • Addition/removal of glucose after cooling affected sperm motility but not viability. • Minimum concentration of added glucose in extender to optimize sperm motility was 40 mM. [ABSTRACT FROM AUTHOR]

It was assessed the effect of the inclusion of Moringa oleifera Lam. and Trichanthera gigantea (Bonpl.) Nees in the diet of hair (Pelibuey) sheep on the quality of their semen. During 90 d, the diets of 15 sheep (24 kg ± 3.95) were divided into three treatments: T1: integral diet with 40 % of M. oleifera + Taiwan grass (Pennisetum purpureum Schumach.), T2: integral diet with 40 % of T. gigantea + Taiwan grass, and T3: 40 % commercial feed + Taiwan grass. The daily weight gain (DWG), carcass yield (CY), testicular development (DT) determined by the diameter (AE) and the scrotal circumference (SC) of these sheep were determined, and the volume, concentration, motility (CASA), viability (SYBR-14/IP), mitochondrial activity (J-C1) and acrosomal integrity (FITC-PSA) of their sperm samples were assessed. No differences were found (P>0.05) in the GDP and RC. Differences were found (P<0.05) in DT, T1 (AE= 48.84 ± 5.99 mm, CE=$9.29 ± 1.13 cm) and T3 (AE= 48.83 ± 4.34 mm, C = 26.62 ± 1.27 cm) exhibited higher values than T2 (44.57 ± 5.59 AE= mm, CE= 25.42 ± 1.50 cm); as for viability, T2 (62.90 ± 6.10 %) and T1 (54.00 ± 6.61 %) had higher percentages than T3 (24.45 ± 7.56 %); in motility, T1 (93.9 ± 2.1 %) and T2 (88.6 ± 1.9 %) had a higher percentage than T3 (71.9 ± 4.0 %). The inclusion of M. oleifera and T. gigantea in the diet allows to obtain a GDP, RC and DT similar to those of commercial feed and increases the number of viable sperm cells by more than 20 %; it also improves certain parameters of motility, thereby improving the reproductive potential of the stallions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Commercially available vaginal lubricants, typically labeled as non-spermicidal, are used to lubricate equine artificial vaginas prior to semen collection. Improper type or amount of lubricant might affect stallion sperm quality, either after short-time exposure or following cooled storage of extended semen previously exposed to lubricant. The aim of this study was to evaluate stallion sperm quality following exposure to lubricant-containing extender for 1 h (T1h) or 24 h (T24h). Three ejaculates were collected from each of four stallions using a small volume of petrolatum to lubricate artificial vaginas, and gel-free semen was diluted to 30 × 106 sperm/mL in extender containing: no lubricant (control), or 1 or 5% (v/v) HR® Lubricating Jelly (HR1 or HR5); K-Y® Jelly (KY1 or KY5); Therio-gel® (TG1 or TG5); Priority Care® Sterile Lubricating Jelly (PC1 or PC5); or Clarity® A.I. Lubricating Jelly (CL1 or CL5). Sperm were evaluated at T1h and T24h for percentages of: total and progressive sperm motility (TMOT and PMOT); curvilinear velocity (VCL; μm/s); and straightness (STR; %); viable acrosome intact sperm (VAI); sperm with abnormal DNA (COMP-α t); viable lipid peroxidation negative sperm (VLPN); and sperm with no detectable DNA oxidative injury [8OHdG(−)]. Following short-term exposure of sperm to lubricants, KY5 reduced TMOT, PMOT, VCL, VAI, VLPN, and COMP-α t in comparison with controls (i.e., P < 0.05). PC5 reduced TMOT, PMOT, VCL, VAI, and 8OHdG(−), and KY1 reduced TMOT, VAI, VLPN in comparison to controls (P < 0.05). Lubricant CL1, HR1 and HR5 yielded similar values to controls for all 8 endpoints, and CL5 yielded similar values to controls for all 8 endpoints (P > 0.05), except for VCL. Following long-term exposure, KY5 decreased TMOT, PMOT, VCL, VAI, VLPN, and COMP-α t as compared to controls (i.e., P < 0.05), PC5 decreased TMOT, VCL, VAI, and 8OHdG(−)as compared to controls in PC5, and KY1 decreased TMOT, VAI, VLPN, and COMP-α t (P < 0.05). TG5 decreased TMOT, PMOT, and VCL as compared to controls (P < 0.05). Lubricant CL5 decreased VCL (P < 0.05), and CL1, HR5, HR1, PC1, and TG1 were similar to controls for all 8 endpoints (P > 0.05). Overall, lubricant KY was the most detrimental to sperm quality, with most profound changes detected at a 5% concentration. Lubricants CL and HR were generally similar to controls and were less affected by lubricant concentration. • Exposure of sperm to lubricants can be detrimental to sperm quality. • Most sperm quality measures decreased following exposure to KY. • Exposure of sperm to CL or HR generally did not affect sperm quality. [ABSTRACT FROM AUTHOR]

We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose–EDTA–egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk–egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially thawed to 5 °C, diluted and refrozen in SMEY containing 2, 4, 6 or 8% GLY or GMF. In Experiment 1, sperm motility was reduced after each cryopreservation cycle (P < 0.05). Extender type did not affect motility after refreezing (P > 0.05), but sperm initially thawed to 5 °C exhibited higher motility than sperm thawed to 37 °C (P < 0.05). In addition, sperm refrozen in SMEY containing MF or GMF exhibited higher motility than sperm refrozen in GLY alone (P < 0.05). In experiment 2, there was an interaction between CPA and CPA concentration (P < 0.05). Sperm refrozen with GMF had higher motility than refrozen sperm with GLY (P < 0.05), and while GLY concentration did not affect post-thaw motility (P > 0.05). Sperm refrozen with 6 or 8% GMF exhibited the highest motility (P < 0.05). In conclusion, sperm motility is best maintained when thawing and refreezing stallion sperm in low sperm concentration ICSI doses by initially thawing the sperm to 5 °C and diluting the sperm in a freezing extender with 8% GMF. • Sperm motility was reduced after the first and second cryopreservation cycle. • Refrozen stallion sperm maintained adequate motility for ICSI selection techniques. • Frozen sperm thawed to 5 °C had higher motility after refreezing than sperm thawed to 37 °C. • Refreezing in a skim milk extender with 8% GMF was better than glycerol. [ABSTRACT FROM AUTHOR]

Background: The population of stallion spermatozoa that survive thawing experience compromised mitochondrial functionality and accelerated senescence, among other changes. It is known that stallion spermatozoa show very active oxidative phosphorylation that may accelerate sperm senescence through increased production of reactive oxygen species. Rosiglitazone has been proven to enhance the glycolytic capability of stallion spermatozoa maintained at ambient temperature. Objectives: Thus, we hypothesized that thawed sperm may also benefit from rosiglitazone supplementation. Materials and methods: Thawed sperm were washed and resuspended in Tyrodes media, and the samples were divided and supplemented with 0 or 75 μM rosiglitazone. After one and two hours of incubation, mitochondrial functionality, Akt phosphorylation and caspase 3 activity were evaluated. Additional samples were incubated in the presence of an Akt1/2 inhibitor, compound C (an AMPK inhibitor) or GW9662 (an antagonist of the PPARγ receptor). Results: Rosiglitazone maintained Akt phosphorylation and reduced caspase 3 activation (p<0.01), both of which were prevented by incubation in the presence of the three inhibitors. Rosiglitazone also enhanced mitochondrial functionality (P<0.01). Conclusion: We provide the first evidence that the functionality of frozen stallion spermatozoa can be potentially improved after thawing through the activation of pro survival pathways, providing new clues for improving current sperm biotechnology. [ABSTRACT FROM AUTHOR]

Semen was collected from the exotic stallions and exotic jacks (n=5 each) using an artificial vagina. The fresh semen was treated with primary extender and centrifuged to remove seminal plasma. Sperm pellet obtained after centrifugation was equally divided to receive secondary extender containing same amount (5%) of different permeable cryoprotectants, either glycerol (Gly) or dimethyl formamide (DMF) before submitting for cryopreservation. Various sperm parameters were assessed at pre-freeze and post-thaw stage which revealed differences (p<0.05) in motility, liveability, plasma membrane integrity and acrosome integrity of stallion as well as Jack semen cryopreserved with DMF or Gly. A variation (p<0.05) existed between individual stallions and jacks extended either with DMF or Gly. In brief, cryoprotectant DMF for stallion and Gly for jack semen were suitable for retaining the plasma and acrosome integrity of spermatozoa. [ABSTRACT FROM AUTHOR]