Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (?Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P‐tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30‐ and 120‐min incubation. Membrane fluidity (MC540) increased in both media at 30‐ and 120‐min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2‐ and 4‐hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation‐related parameters in stallion sperm.     

Cryopreservation of semen from a stallion with seminal vesiculitis

A 6‐year‐old Warmblood stallion was admitted for semen collection and cryopreservation. On the seventh and subsequent collection days semen samples were contaminated with purulent debris. A diagnosis of seminal vesiculitis was made following ultrasonography and endoscopy of the seminal vesicles. The stallion was treated with systemic and topical antimicrobial therapy and, although this did not cure the condition, subsequent ejaculates were suitable for cryopreservation.     

A comparison of duck and chicken egg yolk for cryopreservation of stallion sperm

OBJECTIVE: Duck and chicken egg yolk were compared for their protective effects against cold shock during the cryopreservation of stallion sperm in a lactose-EDTA-glycerol cryodiluent. DESIGN: A completely randomised design was used. Procedure Ejaculates from five stallions (n = 14 ejaculates) were split and diluted to either 20 or 200 x 10(6) sperm/mL in a lactose-EDTA extender containing either duck or chicken egg yolk. The extended semen was then frozen in liquid nitrogen. The percentage of sperm total motility and forward progressive motility were assessed before freezing and at 0 and 1 hr after thawing. Morphology data were also collected at 0 and 1 hr post thaw. RESULTS: Total and forward progressive motility were higher when the sperm were frozen in the presence of duck rather than chicken egg yolk. Furthermore, the total and forward progressive motility and percentage of morphologically normal sperm were higher when frozen at a concentration of 200 than 20 x 10(6)/mL. CONCLUSION: The results of this study demonstrate that the motility parameters of stallion sperm are improved when the semen is frozen in lactose EDTA extender supplemented with duck egg yolk rather than chicken egg yolk. Moreover, sperm motility and the percentage of morphologically normal sperm were higher after freezing at a concentration of 200 x 10(6)/ml rather than 20 x 10(6)/ml.     

Lactoferrin increases sperm membrane functionality of frozen equine semen

During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 μg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome‐reacted sperm were evaluated with a computer‐assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 μM/μg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.     

Cryopreservation of ram semen: Manual versus programmable freezing and different lengths of equilibration

The aim of our study was to examine effects of the length of semen equilibration as well as two freezing techniques on ram sperm post-thaw quality. The ejaculates of Wallachian sheep rams (n = 12) were collected by an electro-ejaculation, equilibrated in a Triladyl® (0, 2, 4, 6, and 8 h) containing glycerol and egg yolk and frozen by programmable freezing (PF) or manual freezing (MF). After thawing, sperm samples were subjected to the motility (computer-assisted sperm analysis [CASA]), viability (SYBR-14/PI), and fertilizing ability (FA) (in vitro penetration/fertilization test on bovine oocytes) assays. It was found that the equilibration of 6 h (E-6) ensured higher post-thaw sperm motility and progressive movement compared with other lengths tested, irrespective of a freezing technique. The E-6 sperm viability did not differ between PF and MF but was lower (P < 0.05) than control. Sperm FA (E-6) was similar in PF (60.44%) and MF (62%) but slightly lower than in fresh (72.8%). Our data demonstrate that the use of MF was comparable with PF, which can be applied in the field conditions without need in a piece of cost-expensive equipment, which can greatly benefit the gene bank of animal genetic resources.     

Use of nutraceuticals in the stallion: Effects on semen quality and preservation

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.     

Cryopreservation of boar semen. II: Effect of cooling rate and duration of freezing point plateau on boar semen frozen in mini- and maxi-straws and plastic bags.

The post-thaw motility and the acrosome integrity of semen from 4 boars frozen with a programmable freezing machine, in mini (0.25 ml) and maxi (5 ml) plastic straws and in 10 x 5 cm Teflon FEP-plastic bags (0.12 mm thick, 5 ml), were compared. The freezing of the semen was monitored by way of thermo-couples placed in the straws and the bags. Three freezing programmes were used, namely A: from +5 degrees C, at a rate of 3 degrees C/min, to -6 degrees C, held for 1 min at -6 degrees C, and followed by a cooling rate of 20 degrees C/min to -100 degrees C; B: a similar curve except that there was no holding time at -6 degrees C and that the cooling rate was 30 degrees C/min, and C: from +5 degrees C to -100 degrees C, with a cooling rate of 35 degrees C/min, followed by storage in liquid N2. Despite the freezing curve assayed, both the mini-straws and the bags depicted much shorter freezing point plateaus as compared to the maxi-straws. Post-thaw sperm motility as well as the amount of normal apical ridges were equally significantly higher when semen was frozen in mini-straws or in bags than in maxi-straws. Significant differences in these post-thawing parameters were obtained between the freezing curves used. The stepwise freezing procedure A appeared as the best alternative for boar semen, considering this in vitro evaluation.     

Comparison of density gradient and single layer centrifugation of stallion spermatozoa: Yield,motility and survival

Reasons for performing study: A new, simpler, technique of colloidal centrifugation has recently been developed, designated single layer centrifugation (SLC). This technique requires evaluation by comparison with a density gradient for its ability to select the best quality spermatozoa and its practicality of use on studfarms. Objective: To compare the effect of 2 methods of colloidal centrifugation, density gradient centrifugation and single layer centrifugation, on stallion sperm motility, yield and survival, using freshly collected extended stallion semen. Methods: Aliquots of extended stallion semen from 10 stallions (38 ejaculates) were processed by the 2 methods of colloidal centrifugation. For both uncentrifuged and centrifuged samples, sperm yield was calculated and subjective sperm motility assessed over several days to provide an estimate of sperm survival. Some stored semen samples, held at 4°C overnight, were also available for testing. Results: For fresh, extended semen, a similar recovery yield of motile spermatozoa was seen for the 2 methods of preparation for single layers and density gradients, respectively. Sperm motility and survival rate were significantly improved by colloidal centrifugation compared to unprocessed ejaculate, without any significant difference between methods (SLC vs. gradient). However, the yield was reduced by 18–20% when cold‐stored semen was used for centrifugation compared to fresh semen; and more variation between ejaculates was observed than for fresh ejaculates. Again, sperm motility and sperm survival were improved in the centrifuged sperm preparations compared to stored, unprocessed ejaculates. Potential relevance: The 2 colloid centrifugation techniques produce equivalent sperm preparations in terms of sperm quality. However, the SLC method would be more practical and convenient for use in the field.     

提高奶牛冷冻精液使用效果的措施

如何提高奶牛冷冻精液的使用效果，在奶牛繁殖中是人们一直追求的目标。根据多年的实际生产经验，在冷冻精液的冷冻方法、解冻液配方、解冻温度、解冻后的保存温度等方面进行了改进，取得了较好的效果。     

Cryopreservation of Black Bengal buck semen: Effects of diluents and freezing on sperm motility and morphology

Semen from Black Bengal bucks was collected to establish a cooling protocol (to −196°C) for buck semen preservation, and to study the effect of freezing on sperm motility and morphology. Semen was diluted with diluents (Triladyl & Tris) and cryoprotectants, filled into straws, sealed, cooled (to 5°C) and equilibrated. After dilution, motility ranged from 75.00% to 76.67% and from 73.33% to 80.00% in Triladyl and Tris diluents, respectively. Motility of sperm after cooling to 5°C in Triladyl and Tris diluents ranged from 65.00% to 66.67% and from 63.33% to 70.00%, respectively. After equilibration in straws, the semen was subjected to a freezing protocol in a computer-controlled biofreezer CL-3000 (cooling at 10°C per minute, from 5°C to −80°C) and plunged into liquid nitrogen. Sperm motility of re-thawed semen varied from 38.33% to 43.33% and from 6.00% to−6.67% in Triladyl and Tris diluents, respectively. Sperm morphology of re-thawed semen was studied and head damage or cryoinjury was found in 2–3% of sperm in Triladyl diluents and 3–6% in Tris diluents. Whether the differences of sperm motility and head damage reflect fertility after artificial insemination is yet unknown and needs to be studied further.     

Comparative effect of slow and rapid freezing on sperm functional attributes and oxidative stress parameters of goat spermatozoa cryopreserved with tiger nut milk extender

Comparative effect of slow and rapid freezing on sperm functional attributes and oxidative stress parameters of goat spermatozoa cryopreserved with tiger nut milk (TNM) extender was examined in this study. Pooled semen samples obtained from West African Dwarf (WAD) goat bucks were diluted with Tris‐based extenders containing different levels of TNM (0, 5, 10, 15 and 20 ml/100 ml extender). The diluted semen samples were subjected to slow and rapid freezing for a period of 7 days and thereafter evaluated for sperm functional attributes (percentage motility, acrosome integrity, membrane integrity, abnormality and livability) and oxidative stress (malondialdehyde [MDA] concentration and acrosin activity) parameters. Results showed that higher (p < 0.05) motility, livability, membrane and acrosome integrities in semen cryopreserved with slow freezing compared to rapid freezing. These parameters (motility, livability and membrane integrity) were higher (p < 0.05) in semen cryopreserved with 15% TNM in both slow and rapid freezing protocols. The results revealed that semen cryopreserved in slow freezing had lower (p < 0.05) abnormality compared to rapid freezing. Acrosin activity was higher in slow freezing compared to rapid freezing. Acrosin activity was higher at 15% TNM in both slow and rapid freezing. Lower (p < 0.05) MDA concentration was observed in semen cryopreserved using slow freezing compared to rapid freezing. The findings revealed improved post‐thaw sperm functional attributes and oxidative stress parameters of WAD goat spermatozoa cryopreserved with 15% TNM using slow freezing.     

Characterization of semen collected by pharmacologically induced ejaculation from a stallion with seminal vesiculitis

The present study compared the quality of sperm collected by artificial vagina or pharmacologically induced ejaculation from a 10-year-old thoroughbred stallion with seminal vesiculitis. The pharmacological protocol involved intravenous administration of detomidine (0.01 mg/kg) and oxytocin (20 IU) and successfully induced ejaculation in all attempts of semen collection. Sperm motility, plasma membrane and acrosome integrity (PMAI), reactive oxygen species (ROS) levels, polymorphonuclear neutrophil (PMN) percentage, and bacterial profiles of fresh and cooled semen (5°C for 24 hr) were evaluated. Semen obtained by the pharmacological method presented reduced seminal volume, decreased PMN percentage and superior sperm motility in cooled samples. Moreover, higher PMAI and lower ROS levels were observed in semen collected by the pharmacological method. Therefore, pharmacologically induced ejaculation is an alternative to obtain semen with minimal contamination and with sperm of superior quality and longevity from stallions with seminal vesiculitis.     

提高奶牛细管冻精精子活力的试验研究

为了提高奶牛细管冻精精子的活力,试验探索了稀释液种类、最佳熏蒸距离、最佳冷冻温度、最佳熏蒸时间、不同解冻温度及时间对精子活力的影响。结果表明:精子在由柠檬酸钠和果糖组成的稀释液中存活时间长,在三羟甲基氨基甲烷稀释液中冷冻后精子活力高于其他稀释液;牛细管冻精最佳熏蒸距离为2.5 cm,时间影响不显著(5～10 min均可);用50℃温水解冻15 s的精子活力比其他解冻温度和时间时的精子活力要好。     

In vitro evaluation of frozen-thawed stallion semen: a review

The article reviews methods used for in vitro evaluation of sperm, with particular emphasis on frozen-thawed stallion sperm. The techniques, limitations of the methods and correlations with fertility results are discussed. Very few studies have tried to find correlation between fertility of frozen stallion semen and laboratory tests. It is difficult and expensive to inseminate an adequate number of mares to achieve statistically significant differences. Significant, but low correlations have been demonstrated between the foaling rate and subjective motility of sperm incubated for 2 h and 4 h at 37 degrees C and hypoosmotic swelling test after 0 and 3 h of incubation. Significant correlations have been reported between the pregnancy rate and viability of propidium iodide-stained sperm assessed by flow cytometry as well as for glass wool and Sephadex filtration tests. No correlations have been detected between fertility and motility immediately after thawing. In spite of that, motility estimation by light microscope is the most commonly used method to evaluate frozen-thawed stallion sperm. Computer assisted automatic sperm analyzers have replaced light microscopy in research projects, but so far nobody has been able to demonstrate a correlation between fertility of frozen stallion semen and any of the motility parameters obtained by these instruments.     

Effects of two freezing methods and two cryopreservation media on post‐thaw quality of stallion spermatozoa

Glycerol‐based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post‐thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol‐based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio^®). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio^® in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio^® in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio^® using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio^® (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio^® using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.     

Imaging flow cytometry to characterize the relationship between abnormal sperm morphologies and reactive oxygen species in stallion sperm

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.