Stallion Sperm Research Articles

A matter of agreement: The effect of the technique and evaluator on the analysis of morphologic defects in stallion sperm.

Analysis of sperm morphology is an important part of the stallion breeding soundness evaluation since it provides an objective measure of a stallion's sperm quality and is one of many factors that estimate a stallion's fertility potential. To describe the effect of sperm quality level on the technique (Differential Interference Contrast - DIC; Phase-contrast - PH; Dip-Quick staining - DQ; and eosin-nigrosin staining - EN; semen samples fixed in buffered-formal saline) and evaluator (three evaluators; using only DIC), stallions were categorized based on sperm quality into three categories: High: >57% normal sperm, Moderate: 23-56% normal sperm, or Low: <23% normal sperm (four stallions per category). The data were analyzed using three different statistical methods: Analysis of Variance (ANOVA), correlative analysis, and Bland-Altman method (agreement). A higher level of agreement among techniques was observed between DIC and PH for morphologically normal sperm. The agreement between the alternative methods (EN, DQ, or PH) and the standard method (DIC) varied, depending on the sperm quality level (High, Moderate, or Low). Some morphological defects (e.g., AH, AMP) were constantly underestimated with the staining methods (DQ, EN) compared to DIC and PH, particularly in ejaculates with low sperm morphology. Underestimation of some abnormalities, due to the technique or the evaluator, has the potential to alter the clinical interpretation of stallion fertility.

Effect of Isolation Protocols and Cryoprotectants on Freezing of Stallion Epididymal Spermatozoa

BACKGROUND: The recovery of spermatozoa from the cauda epididymis may be the only option to obtain genetic material from elite stallions that had undergone castration or sudden death due to colic or severe injury. OBJECTIVE: To evaluate two different protocols for retrieval of stallion epididymal spermatozoa and to evaluate different cryoprotectants on the freezability of the epididymal spermatozoa. MATERIALS AND METHODS: Six epididymides from three stallions were collected immediately after routine castration under general anesthesia. In the first experiment, each epididymis (of two testes) of the same stallion were processed using different methods for retrieval of the epididymal spermatozoa and were pooled and cryopreserved either using 5% glycerol or 5% dimethyl formamide (DMF) as cryoprotectant. The semen quality parameters viz., progressive motility, HOST, viability and acrosome integrity were evaluated at the fresh, pre-freeze and post-thaw stages. RESULTS:Retrograde method of flushing of epididymis yielded significantly (ρ &lt; 0.05) higher concentration of the stallion sperm than that of the floating method. The qualitative semen parameters i. e., viability, plasma membrane integrity and acrosome integrity were found to be significantly restored using 5% DMF as cryoprotectant in comparison to when 5% glycerol was used. CONCLUSION: Retrograde flushing method of epididymis yielded significantly higher sperm concentration to that of the floating method, and 5% DMF as cryoprotectant provided acceptable freezability of stallion epididymal spermatozoa.

Carboxymethylchitosan with medium-molecular-weight affects kinectics and acrosome of stallion sperm after freezing/thawing

Semen cryopreservation ensures the storage of stallion genetics for an unlimited time. The improvement of extenders with new antioxidant substances can optimize the properties of post-thawed semen. The study aimed to investigate the addition effect of medium-molecular-weight carboxymethylchitosan (CQm) derivates to freezing diluent of stallion sperm after freezinf/thawing. Twice a week, five ejaculates of four stallions were obtained, totalizing 20 ejaculates. Semen was diluted in commercial freezing extender (Botucrio) supplemented with CQm: control (0), 0.75, 1.5, and 3 mg/mL. Samples were filled in straws (0.5 mL) and submitted to freezing and storage at -196°C. Thawing was performed at 37°C/30 s, and the samples of each group were analyzed for kinetics, plasma membrane integrity, acrosome membrane integrity, and mitochondrial membrane potential . The addition of 1.5 and 3 mg/mL CQm showed lower values (P < .05) of total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP) and wobble (WOB), comparing to control group. Besides, it was observed lower (P < .05) percentages of sperm with intact acrosomes in the group treated with 3 mg/mL of CQm than control group. In conclusion, high concentration of medium-molecular-weight carboxymethylchitosan to freezing diluent damages kinematic and acrosome of stallion sperm after freezing/thawing.

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

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

Egg yolk plasma enriched with β-carotene through the diet of laying hens and adding it to the extender improves the quality of frozen semen in Arabic stallions.

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 1.4nmol/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.

Deciphering sperm chromatin properties to predict stallion sperm fertility

Although previous studies have examined the relationship between the sperm DNA fragmentation index and fertility in stallions, other aspects of chromatin structure or packaging and fertility have not been explored. In the present study, relationships between fertility and DNA fragmentation index, protamine deficiency, total thiols, free thiols and disulfide bonds in stallion spermatozoa were investigated. Ejaculates (n = 36) were collected from 12 stallions and extended to prepare semen doses for insemination. One dose from each ejaculate was sent to the Swedish University of Agricultural Sciences. Aliquots of semen were stained for flow cytometry with acridine orange for the Sperm Chromatin Structure Assay (DNA fragmentation Index, %DFI), with chromomycin A3 (CMA) for protamine deficiency, and with monobromobimane (mBBr) for detection of total and free thiols and disulfide bonds. Per season pregnancy rates after insemination were obtained. Mixed linear models were used to analyze data. Negative correlations were found between pregnancy rate and %DFI (r = −0.35, P < 0.03) and pregnancy rate and free thiols (r = −0.60, P < 0.0001). Furthermore, there were positive correlations between total thiols and disulfide bonds (r = 0.95, P < 0.0001), and protamine and disulfide bonds (r = 0.4100, P < 0.01986). Since chromatin integrity, protamine deficiency and packaging were all associated with fertility, a combination of these factors could be used as a biomarker of fertility when assessing ejaculates.

A Combination of Taurine and Caffeine in Stallion Semen Extender Positively Affects the Spermatozoa Parameters.

This study was aimed to determine the impact of different taurine and caffeine combinations on the motility, viability, and oxidative markers of chilled stallion spermatozoa. Each stallion semen sample was diluted in a ratio of 1:2, with various taurine and caffeine concentrations (2.5-7.5 mg/mL taurine + 0.625-1.25 mg/mL caffeine) dissolved in a conventional extender. The control samples (CON) were prepared by diluting ejaculate only using the conventional extender. The motility was analyzed using a CASA system at different time intervals (0, 6, 12, 24, and 30 h) and the viability was evaluated using a mitochondrial toxicity test (MTT) performed at the end of the incubation at 5 °C. The liquid part of experimental samples was separated by centrifugation after 30 h of incubation and underwent the evaluation of oxidative stress via the quantification of markers ferric reducing ability of plasma (FRAP) and total oxidant status (TOS). The samples that were treated with a combination of taurine and caffeine significantly improved the motility parameters, mainly after 12, 24, and 30 h of incubation. Samples extended with combination of taurine and caffeine neither compromise viability nor alterations of redox status. The results of this study describe the combination of taurine and caffeine as an optimal supplement for improving the quality of stallion semen during chilled storage.

The Autophagy Marker LC3 Is Processed during the Sperm Capacitation and the Acrosome Reaction and Translocates to the Acrosome Where It Colocalizes with the Acrosomal Membranes in Horse Spermatozoa

Despite its importance in somatic cells and during spermatogenesis, little is known about the role that autophagy may play in ejaculated spermatozoa. Our aim was to investigate whether the molecular components of autophagy, such as microtubule-associated protein 1 light chain 3 (LC3), are activated in stallion spermatozoa during the capacitation and acrosome reaction and if this activation could modulate these biological processes. To analyze the autophagy turnover, LC3I and LC3II proteins were assessed by western blotting, and the ratio between both proteins (LC3II/LC3I) was calculated. In somatic cells, this ratio indicates that autophagy has been activated and similar LC3 processing has been described in mammalian spermatozoa. The subcellular localization of autophagy-related proteins was assessed by immunofluorescence with specific antibodies that recognized Atg16, Beclin-1, and LC3. The colocalization of acrosomal membranes (PNA) and LC3 was studied by confocal microcopy, and the acrosome reacted cells were quantified by flow cytometry. The incubation of stallion sperm in capacitating conditions (BWW; 3 h) significantly increased LC3 processing. This increment was three to four times higher after the induction of the acrosome reaction in these cells. LC3 was mainly expressed in the head in mature ejaculated sperm showing a clear redistribution from the post-acrosomal region to the acrosome upon the incubation of sperm in capacitating conditions (BWW, 3 h). After the induction of the acrosome reaction, LC3 colocalized with the acrosome or the apical plasmalemma membranes in the head of the stallion spermatozoa. The inhibition or activation of autophagy-related pathways in the presence of autophagy activators (STF-62247) or inhibitors (E-64d, chloroquine) significantly increased LC3 processing and increased the percent of acrosome reacted cells, whereas 3-methyladenine almost completely inhibited LC3 processing and the acrosome reaction. In conclusion, we found that sperm capacitation and acrosome reaction could be regulated by autophagy components in sperm cells ex vivo by processes that might be independent of the intraluminal pH of the acrosome and dependent of LC3 lipidation. It can be speculated that, in stallion sperm, a form of noncanonical autophagy utilizes some components of autophagy machinery to facilitate the acrosome reaction.

A Pilot Study on the Uptake of Propidium Iodide and YO-PRO-1 Iodide through the Pannexin Channels in Wallachian Frozen-Thawed Ram Spermatozoa.

Propidium iodide (PI) and YO-PRO-1 (YPI) dyes are routinely used to determine sperm viability in many livestock species. It is commonly accepted that these dyes penetrate only sperm cells with damaged plasma membranes. Recently, however, the mechanism of dye uptake unrelated to damaged plasma membranes, but instead related to pannexin channels in dog and stallion sperm cells was demonstrated. This pilot study aimed to evaluate the role of pannexins in the uptake of PI and YPI dyes on Wallachian frozen-thawed ram spermatozoa by flow cytometry using probenecid, a specific inhibitor of pannexin channels. Additionally, the expression of pannexins in Wallachian sperm was evaluated directly (by qRT-PCR). The results demonstrate the active role of pannexin channels in the uptake of PI and YPI dyes on frozen-thawed Wallachian ram sperm. In conclusion, when using the PI or YPI exclusion assay to determine Wallachian frozen-thawed ram sperm viability, the danger of overestimating the number of spermatozoa with the damaged plasma membrane must be considered. The observed breed-specific, and more importantly, individual differences in gene expression as well as in dye uptake indicate the need for further studies.

The effect of serum lipids and spermoplasm on the stallion sperm quality

The quality and cryoresistance of stallion sperm depend on many endogenous and exogenous factors. One of the factors affecting the sperm quality are the parameters of the lipid spectrum of spermoplasm and blood serum. Lipase activity, cholesterol and triglyceride content in blood serum and spermoplasm were studied in 19 stallions of the Arabian breed aged from 5 years to 15 years (on average 10.1±0.3 years). It was found that lipase activity in the spermoplasm (217.0 U/l) is on average 11.4 times higher than in the blood serum (19.1 U/l). The concentration of spermoplasm triglycerides (1.3 mmol/l) is on average 4.3 times higher than in blood serum (0.3 mmol/l). The concentration of cholesterol in the blood serum (2.4 mmol/ l) is on average 12 times higher than the same indicator in the stallion spermoplasm (0.2 mmol/l). The relationship between the parameters of the lipid spectrum of blood serum and spermoplasm with the characteristics of stallion sperm was studied. A significant relationship was found between the activity of lipase in blood serum with progressive mobility (r=0.66; p=0.006) and survival (r=0.67; p=0.005) of spermatozoa in cryopreserved sperm.

PROTECTIVE ENVIRONMENTS FOR ANIMAL SPERM: EVOLUTION OF METHODS AND TOPICAL ASPECTS (REVIEW ARTICLE)

A retrospective review of domestic and foreign sources of literature is presented, as well as data of published own research on cryopreservation of animal sperm. The main historical stages of the creation of protective environments for deep freezing of sperm are given. In the 30s of the last century, a phenomenon characterized by the death of spermatozoa upon sharp cooling in the range of positive temperatures was discovered. It is called temperature shock of sperm. To prevent it, it is proposed to add substances containing phospholipids to the composition of diluents. Such environments can contain both simple components - native chicken egg yolk or milk, and high-tech - lipoproteins, isolated phospholipids of various origins. To stabilize protein-lipid complexes of plasma membranes and acrosomes of sperm during the cooling process, carbohydrates are added to the diluents. Sugars are components of energy supply for sperm and, along with salts, they are the main osmotic regulators. A combination of two or three carbohydrates in the medium was traditionally considered necessary. However, the Kharkiv school of reproductive specialists has proven the possibility of creating effective protective environments using only one sugar - sucrose or lactose - based on considerable practical experience. The effectiveness of germ cell freezing is shown depending on the cryoprotectants used. Glycerin is the first known endocellular cryoprotectant, which is still unsurpassed in sperm cryopreservation. Our own experimental data on the effect of combinations of glycerol with substances from the amide group on the main biological indicators of sperm after deconservation are presented. Cryoprotectants dimethylacetamide (DMAC) and dimethylformamide (DMF) were tested in own experiments on stallion semen. The experiments studied the effect of different concentrations of the above-mentioned penetrating cryoprotectants both on the main physiological characteristics of stallion sperm (motility, survival), and on the degree of damage to the membrane apparatus of sperm. The effectiveness of certain combinations of these substances has been proven. Methods of preventing the negative impact of oxygen and the development of lipid peroxidation processes in sperm during cryopreservation are presented. The concept of using additional hormonal components in diluents, in particular prostaglandin F2a, is revealed. The materials related to the effect on the quality of reproductive cells of healing preparations are displayed. Keywords: artificial insemination, environments, semen, animals, bulls, stallions, cryoprotectants, freezing.

In vitro aging of stallion spermatozoa during prolonged storage at 5°C.

Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca2+ ]i and plasma membrane fluidity in viable, acrosome-intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5°C. High proportions of viable and acrosome-intact spermatozoa (71 ± 8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was well preserved (n=17 stallions). In addition, more than 90% of viable, acrosome-intact spermatozoa had active mitochondria and low intra-cellular or mitochondrial ROS levels. By contrast, the percentage of viable, acrosome-intact sperm with low plasma membrane fluidity and low [Ca2+ ]i decreased over time (1h: 63 ± 16%, 96 h: 29 ± 18%; p < 0.05). The [Ca2+ ]i in viable sperm rose 3.1-fold (p < 0.05) over the 4 days, and fewer spermatozoa responded to bicarbonate stimulation (1h: 46 ± 17%, 96 h: 19 ± 12%) with an increase in plasma membrane fluidity following prolonged storage. Overall, prolonged storage of stallion semen at 5°C resulted in disturbed calcium homeostasis and increased plasma membrane fluidity. The decline in fertility of stallion semen during cooled-storage may therefore relate to aspects of in vitro aging (changes in plasma membrane fluidity and intracellular calcium) which impairs capacitation-associated cell functions.

The level of acute phase blood proteins as a predictive criterion of the cryo resistance of stallion sperm

The indicators of the biochemical analysis of the blood of horses reflect the state of metabolism of the animal and at the same time are potential predictors of the qualitative characteristics of spermatozoa, the freezing of which is one of the most important ways to preserve and further use the genetic material. The aim of this study was to identify correlations between the level of such acute phase blood proteins, as C-reactive protein and fibrinogen, and the qualitative and quantitative characteristics of the native and thawed sperm of stallions. The object of the study was blood and sperm samples from 87 stallions of various breeds, the average age of which was 10.5±0.5 years. To assess the protein status of the blood, in addition to the aforementioned acute phase proteins, the total protein content, the levels of albumin, globulins, and the albumin / globulin ratio were determined. Stallion sperm samples were assessed according to the following indicators: ejaculate volume, concentration, the total number of spermatozoa in the ejaculate, progressive motility and survival at +4°C. When analyzing the correlations between the studied parameters, a statistically significant negative inverse relationship was revealed between the level of fibrinogen in the blood serum and such parameters as the content of progressively motile sperm in native and thawed cryopreserved sperm (r=-0.22, p&lt;0.05 and r=-0.29, p&lt;0.01, respectively), as well as sperm survival in native and thawed sperm (r=-0.48, p&lt;0.001 and r=-0.22, p&lt;0.05, respectively).There was no statistically significant relationship between the level of C-reactive protein and the studied characteristics of the spermogram. The revealed negative correlation between the level of fibrinogen and the characteristics of the stallions’ sperm can be considered as one of the prognostic important indicators that must be taken into account when selecting stallions with the highest indicators of the quality of cryopreserved sperm.

Phosphoproteomics for the identification of new mechanisms of cryodamage: the role of SPATA18 in the control of stallion sperm function†.

Although recent research has addressed the impact of cryopreservation on the stallion sperm proteome, studies addressing the stallion sperm phosphoproteome are lacking. In the present study, the data set of proteomes of fresh and cryopreserved spermatozoa were reanalyzed, showing that cryopreservation caused significant changes in the phosphoproteome. The phosphoproteins reduced most significantly by cryopreservation were Ca2+binding tyrosine phosphorylation regulated, protein kinase cAMP-activated catalytic subunit beta (CABYR), mitochondria eating protein (SPATA18), A kinase anchoring protein 4 (AKAP4), A-kinase anchoring protein 3 (AKAP3) and the Family with sequence similarity 71 member B (FAM71B). These proteins belong to the gene ontology (GO) terms sperm fibrous sheath (GO: 0035686), and sperm principal piece (GO: 0097228). The regulatory interactions between kinases and phosphorylation sites on the proteins that were affected most were also investigated, and the potential kinases (based on human orthologs) involved in the regulation of these phosphoproteins identified were: PKCß for SPATA18 and GSK3ß for CABYR. Kinase inhibition assays were also conducted showing that kinases phosphorylating the above-mentioned proteins play an important role in their activity and thus, phosphorylation controls the activity of these proteins and their role in the regulation of the functionality and viability of stallion spermatozoa. In conclusion, the data reported here contribute to the understanding of the fact that the dephosphorylation of certain proteins is a molecular lesion induced by cryopreservation in the stallion spermatozoa.

48 Semiquantitative and quantitative assessments of phospholipase C zeta 1 in stallion sperm

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

46 Fertilising ability of Arabian stallion sperm frozen with different cryoprotective agents by heterologous IVF

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

An Investigation of Equine Sperm Quality Following Cryopreservation at Low Sperm Concentration and Repeated Freeze-Thawing

Stallion spermatozoa are typically cryopreserved at 200 to 300 million sperm/ml; however recent advances such as intracytoplasmic sperm injection (ICSI) requires only one spermatozoon, wasting many, after thawing a whole straw. Cryopreserving at concentrations less than the current standard or refreezing thawed spermatozoa could maximize the use of genetically valuable animals and reduce waste. This investigation aimed to identify if lowering the sperm concentration for cryopreservation affected post-thaw quality after one and two freeze-thaw cycles. Nine ejaculates were collected from three fertile, “good freezer” stallions (post-thaw motility ≥35%) for experiment 1. Each ejaculate was split into eight treatments: five, 10, 20, 50, 100, 200, 300, 400 million sperm/ml and cryopreserved. Post-thaw: motility, viability, acrosome integrity and oxidative stress were assessed. Experiment 2, straws from experiment 1 (300 million sperm/ml) were thawed, diluted to 20 million sperm/ml or left undiluted (control) and refrozen. Post-thaw motility and viability were assessed. In experiment 1 sperm concentration did not affect post-thaw total motility (TM), progressive motility (PM) or viability at 50 to 400 million sperm/ml (P > .05). Whilst sperm concentrations of five to 20 million/ml did differ (post-thaw TM and PM). Both refreezing and reducing spermatozoa concentration, decreased TM, PM and viability (P < .05) after two freeze-thaw cycles. These results suggest cryopreserving at sperm concentrations as low as 50 million/ml maintains spermatozoa quality in good freezer stallions. Spermatozoa maintained some motility and viability when initially cryopreserved at 20 million sperm/ml and after two freeze-thaw cycles but research should investigate more optimal conditions.

L-carnitine and acetyl-L-carnitine enhance stallion sperm quality during semen storage at 5°C

Carnitine, a powerful antioxidant, has an essential role in sperm energy metabolism. Among carnitines, only L-carnitine’s effect on stallion semen has been tested and not acetyl-L-Carnitine. Therefore, we aimed to determine the ideal concentrations of L-carnitine (LC) and acetyl-L-carnitine (AC) and their effects on stallion semen cooled at 5℃ for up to 48 hours. Semen was extended to 50 x 106 sperm/ml in commercial extender (Control), and concentrations of 5, 10, and 15 mmol/l of LC and AC were evaluated in Experiment 1. Sperm motility and plasma membrane integrity were assessed by CASA and epifluorescence microscopy, respectively. In Experiment 2, the combination of the intermediate doses of LC (10 mmol/l) and AC (10 mmol/l) was tested. Sperm parameters were evaluated as in Experiment 1 and in addition, DNA fragmentation index (DFI), production of reactive oxygen species (ROS), and lipid peroxidation (PEROX) were evaluated by flow cytometry. All analyses were performed at 0, 24, and 48 hours after semen collection, processing, and cooled-stored at 5°C. In Experiments 1 and 2, the groups supplemented with LC and AC or LC+AC had higher plasma membrane integrity and motility parameters compared to Control group (p &lt; 0.05). The LC and AC combination did not change sperm parameters compared to LC or AC alone (p &gt; 0.05). No differences (p &gt; 0.05) were observed for DFI, ROS, and PEROX. In conclusion, LC and AC’s addition, alone or in combination, enhanced sperm motility and plasma membrane integrity of stallion sperm after cooled-storage at 5℃ for up to 48 hours.

Effects of egg yolk level, penetrating cryoprotectant, and pre-freeze cooling rate, on the post-thaw quality of stallion sperm

The current study determined the effect of the egg-yolk (phospholipid source) level (egg yolk [20% EY] vs. skim-milk + egg yolk [SM + 2% EY]), cryoprotectant (glycerol [Gly] vs. glycerol + methylformamide [Gly + MF]), and pre-freeze cooling rate (–0.1 vs. –1 vs. −5 °C/min) on post-thaw stallion sperm quality. In Experiment 1, ejaculates (n = 27) from 9 stallions (3 ejaculates each) with varied sperm quality (High, Average, or Low) were frozen in EY-Gly, SMEY-Gly, EY-Gly + MF, or SMEY-Gly + MF extenders. Sperm in each group were cooled from 22° to 5°C using either −0.1 °C/min or −1 °C/min linear cooling rates prior to freezing. In Experiment 2, ejaculates (n = 24) from 12 stallions (2 ejaculates each) with High or Average sperm quality were frozen in EY-Gly, EY-Gly + MF, or in BotuCrio (BC) extenders. Sperm in each group were cooled from 22° to 5°C using either –1 or −5 °C/min linear cooling rates prior to freezing. In Experiment 1, for stallions with High or Average sperm quality, either cooling rate generally resulted in lower sperm quality for the SMEY-based extenders than for the EY-based extenders (P < 0.05). Stallions with Low sperm quality were unaffected by any experimental treatment (P > 0.05). In Experiment 2, a −5 °C/min cooling rate yielded lower sperm quality in BC than in EY-Gly or EY-Gly + MF groups (P < 0.05); however, a −1 °C/min cooling rate yielded similar sperm quality among these treatments (P > 0.05). In summary, the phospholipid level in the freezing extender and the pre-freeze cooling rate, but not the penetrating cryoprotectant, affected the post-thaw quality of stallion sperm.

Influences of dietary supplementation with evening primrose oil (Oenothera biennis) as a source of omega-6, linoleic and gamma linolenic acid on the quality and function variables of stallion spermatozoa

Influences of dietary supplementation with evening primrose oil (Oenothera biennis) as a source of omega-6, linoleic and gamma linolenic acid on the quality and function variables of stallion spermatozoa