Effects of cysteine and ergothioneine on post-thawed Merino ram sperm and biochemical parameters

The aim of the current study was to evaluate the effects of cysteine and ergothioneine on the post-thawed sperm parameters, lipid peroxidation and antioxidant activities. Semen samples from 5 mature Merino rams were used in the study. Semen samples, which were diluted with a Tris-based extender containing l-Cysteine and l-(+)-Ergothioneine and no antioxidant (control), were cooled to 5 °C and frozen in 0.25 ml French straws. Frozen straws were then thawed individually at 37 °C for 20 s in a water bath for evaluation.Ergothioneine at doses of 2 and 4 mM increased percentages of subjective motility, VSL and VCL, compared to controls following the freeze-thawing (P < 0.001). Ergothioneine at three different doses led to higher rates of progressive motility and VAP, compared to control groups (P < 0.001). Cysteine and ergothioneine at three doses provided the higher rates of ALH, in comparison to no antioxidant group (P < 0.001). As regards CASA motility, supplementation with antioxidants did not provide any significant difference on the percentage of post-thaw sperm CASA motilities, in comparison to the control. In regards of sperm membrane integrity, only cysteine 1 mM provided a greater protective effect, compared to control (P < 0.001). Percentages of sperm with high mitochondrial activity were dramatically increased with cysteine at doses of 1 and 2 mM, compared to control (P < 0.05). No significant differences were observed in sperm acrosome integrities among groups. CAT activity was increased significantly only in cysteine1 mM compared to control group (P < 0.001). Cysteine at doses of 2 and 4 mM showed a tendency of increased activities of CAT when compared to control. But these increases were not statistically significant. Supplementation with antioxidants did not significantly affect activities of SOD and GPx. Findings of this study showed that ergothioneine supplementation in semen extenders, was of greater benefit to motility and motion characteristics of frozen-thawed ram sperm.     

Methionine supplementation improves ram sperm parameters during liquid storage at 5 °C

The aim of this study was to investigate the effects of methionine and lipoic acid on ram sperm parameters during liquid storage (5 °C). Ejaculates collected from five Merino rams were pooled at 37 °C. Each pooled ejaculate was divided into five equal aliquots and diluted (37 °C) with five extenders, one of which was without additives, two of which contained methionine at two different doses, and the other two of which contained lipoic acid at two different doses. Sperm parameters were determined at 0, 24, 48, 72 and 96 h of liquid storage at 5 °C.     

The synergistic effect of trehalose and low concentrations of cryoprotectants can improve post-thaw ram sperm parameters

The objective of this study was to compare the effects of different concentrations of two different cryoprotectants (glycerol, G and ethylene glycol, EG) and trehalose (T), added to the semen extender, on post-thaw ram sperm parameters. Ejaculates, collected from 6 Merino rams, were pooled and evaluated at 37 °C. The pooled samples were divided into six equal aliquots, and diluted in Tris-based extenders containing 5% G, 3% G + 60 Mm T, 1.5% G + 100 Mm T, 5% EG, 3% EG + 60 mM T, and 1.5% EG + 100 Mm T. Subsequently, the samples were cooled to 5 °C, frozen in 0.25-ml French straws, and stored in liquid nitrogen (LN₂). Frozen samples were thawed individually, at 37 °C for 25 s in a water bath, for evaluation. Sperm motility was assessed using a phase-contrast microscope with a warm stage maintained at 37 °C. Acrosome integrity (FITC/PNA-PI), sperm viability (SYBR-14/PI), mitochondrial activity (JC-1/PI), DNA damage (COMET assay) and DNA fragmentation (TUNEL test) were determined. The group of samples diluted in an extender containing 5% of glycerol (Group 5% G) displayed higher percentages of subjective motility, viability and mitochondrial activity of sperm, compared to the other groups (P < 0.05). On the other hand, Group 3% G + 60 mM T yielded the second-best results for subjective motility, viability and mitochondrial activity of sperm, when compared to the other groups. The post-thaw sperm parameters of Group 3% G + 60 Mm T did not show any statistically significant difference from those of Group 5% G. There were no statistically significant differences between the groups for acrosome integrity (P > 0.05).The results of the COMET assay showed that the use of low concentrations of cryoprotectants in combination with trehalose decreased sperm DNA damage. Accordingly, Group 1.5% G + 100 mM T and Group 3% EG + 60 mM T benefited from a significantly stronger cryoprotective effect on DNA integrity, in comparison to Group 5% G (P < 0.05). According to the results of the TUNEL test, the combined use of low concentrations of cryoprotectants with trehalose decreased sperm DNA damage, when compared to the use of 5% glycerol, but this difference was statistically insignificant (P > 0.05).In conclusion, G and EG concentrations can be reduced by adding various amounts of T (60 mM, 100 mM) to the semen extender. The addition of 5% of glycerol and 3% G + 60 mM T to the semen extender did not yield statistically different post-thaw sperm parameters, when compared for protection against cryoinjury. Post-thaw sperm parameters can be improved by the supplementation of the semen extender with 3% G + 60 mM T. Thus, we recommend the use of freezing extenders containing low cryoprotectant concentrations (3% G) combined with trehalose to avoid the high level of toxic and osmotic damage caused by 5% G.     

Evaluation of ram semen quality using polyacrylamide gel instead of cervical mucus in the sperm penetration test

Fertility is a very complex biological function that depends on several properties of the spermatozoa, including sperm motility. Two objectives are analyzed in this study: (1) Replace the cervical mucus by a synthetic medium in a sperm penetration test, and (2) evaluating the results of this test objectively analyzing the sperm number that migrates. In experiment 1, we have tested eight concentrations of acrylamide (1%-2%). Rheological properties of media were analyzed. The plastic straws, loaded with acrylamide, were placed vertically on the semen sample tube for 15 min at 39 °C. After, the acrylamides were placed, by segments of 5 mm, into wells of a 24-well plate, dyed with Hoechst 33342 and the number of spermatozoa were calculated by automated microscopy analysis. The 1.55% and 1.6% acrylamide gel showed a number of spermatozoa emigrating closer to that seen with natural mucus. In experiment 2, we applied the sperm penetration in acrylamide 1.6% and 1.55% using fresh semen and cooled semen at 15 °C and 5 °C. The spermatozoa counts were performed for each segment of 10 mm. Semen chilled at 15 °C presented intermediate values of sperm counts in comparison with fresh semen (higher) and 5 °C chilled semen. The sperm counts do not differ between acrylamides but the rheological properties of acrylamide 1.6% were more similar to those of the natural cervical mucus. In experiment 3, we have observed significant correlations between the number of spermatozoa and several sperm quality parameters (positive: progressive motility and velocity according to the straight path; negative: damaged acrosomes and apoptotic cells) in 1.6% acrylamide media. We conclude that the size of the cell subpopulation, objectively calculated, that migrate beyond 20 mm in 0.5-mL straws filled with acrylamide is a useful parameter in ram sperm quality assessment and further studies are needed to evaluate its relationship with field fertility.     

Effects of reduced glutathione and catalase on the kinematics and membrane functionality of sperm during liquid storage of ram semen

The objective of this study was to evaluate the effects of reduced glutathione (GSH) and catalase (CAT) supplementation on the kinematics and membrane functionality of sperm during the liquid storage of ram semen, cooled at 5 °C, for up to 24 h. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control) or supplemented with either CAT (100, 200, and 400 U/mL) or GSH (100, 200, and 400 mM) at a final concentration of 50 × 10⁶ sperm/mL. Sperm kinematics, which was analyzed by computer-assisted sperm analysis (CASA), and membrane functionality, which was analyzed using the hypo-osmotic swelling test (HOST), were determined after the addition of the semen samples at different processing times (fresh/diluted, 1.5, 6, 12, and 24 h, at 5 °C). No significant differences were recorded in the kinematics or membrane functionality between treatments at different times. The supplementation of diluents with 100 and 200 U/mL of CAT prevented the harmful effects of cooling on total sperm motility. No significant differences were observed in progressive sperm motility throughout processing, regardless of the treatment and time of evaluation. Supplementation with 400 mM GSH resulted in an earlier reduction (P < 0.05) of total sperm motility, a decrease in rapid sperm rate and a reduction in curvilinear velocity during incubation, at 5 °C. The cooling induced a reduction (P < 0.05) in the percentage of sperm with a functional plasma membrane (HOST), especially after 1.5 h of incubation. Based on the results of the present study, the addition of CAT (100 and 200 U/mL) reduced the deleterious effects of cooling on total motility in ram sperm maintained at 5 °C for 24 h, although it did not affect the functionality of the sperm membranes. However, the addition of 400 mM GSH caused negative effects on the velocity parameters of the sperm.     

Seasonal dynamics of sperm morphometric subpopulations and its association with sperm quality parameters in ram ejaculates

Sperm morphologic assessment is considered an irreplaceable part of standard laboratory routine analyses in the diagnosis of male fertility. Thus, in an attempt to quantify the effects of season on sperm morphology and its functional significance in relation to sperm quality parameters, sperm head morphometric traits were analyzed by using an objective computerized analysis combined with principal components analysis (PCA) cluster analysis to establish the relationship between the distribution of the subpopulations found and sperm quality in each season. There were slight variations on sperm motility and sperm membrane integrity indexes (P > 0.05). However, the mean values for sperm concentration substantially changed among seasons in all individuals studied (P < 0.01). There were significant differences in sperm morphometric parameters (P < 0.01) as well as in the distribution of morphometric subpopulations between seasons (P < 0.001). In conclusion, this study confirmed that there was an important seasonal effect on sperm morphometric traits. In addition, the distribution of these subpopulations seems to be related to the season studied and the ejaculate quality which would be a very important indicator of sperm function. The substantial information derived from these morphometric subpopulations has provided new knowledge which can be used in future studies using sperm morphometry as a seasonal indicator in ram ejaculates.     

Motility and acrosomal integrity comparisons between electro-ejaculated and epididymal ram sperm after exposure to a range of anisosmotic solutions, cryoprotective agents and low temperatures

Effective ram sperm cryopreservation protocols, which would yield acceptable lambing rates following artificial insemination (AI), are currently lacking. The objectives of the current studies were to compare the effects of various anisosmotic conditions, cryoprotective agents (CPAs) and chilling on the motility and acrosomal integrity of electro-ejaculated and epididymal ram sperm. Three experiments were conducted. In experiment 1, ejaculated and epididymal ram sperm were exposed to 75, 150, 225, 600, 900 and 1200 milliosmolal (mOsm)/kg sucrose solutions, held for 5 min and then returned to isosmotic condition. Motility characteristics of sperm during exposure to each anisosmotic solutions and after returning to isosmotic conditions were determined. In experiment 2, ejaculated and epididymal ram sperm were exposed to 1 M glycerol (Gly), dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) for 5 min and then returned to isosmotic conditions. Motility characteristics of sperm samples during exposure to each CPA solution and after returning to isosmotic conditions were determined. In experiment 3, effects of various temperatures on motility characteristics of ejaculated and epididymal ram sperm were determined after exposing them to three different sub-physiologic temperatures (4, 10 and 22 °C) for 30 min and subsequently returning them to 37 °C. The motility of ejaculated ram sperm was significantly more affected from anisosmotic stress than was epididymal ram sperm (P < 0.05). While anisosmotic stress had no effects on acrosomal integrity of epididymal ram sperm, there was a significant reduction in acrosomal integrity for ejaculated ram sperm after the addition and removal of a 75 mOsm sucrose solution. The abrupt addition and removal of 1 M Gly, DMSO, EG or PG had no effect on the motility and acrosomal integrity of epididymal ram sperm (P > 0.05). However, there was a slight decrease in acrosomal integrity for ejaculated ram sperm after exposure to 1 M Gly, DMSO or EG (P > 0.05). Both epididymal and ejaculated ram sperm exhibited temperature-dependent loss of motility and acrosomal integrity (P < 0.05). However, ejaculated ram sperm was more sensitive to chilling stress than epididymal sperm (P < 0.05). In conclusion, the current data suggest that while epididymal ram sperm is extremely resilient to various cryobiologically relevant stress conditions, ejaculated ram sperm demonstrate greater sensitivity to such stressors. These findings should be taken into account when developing cryopreservation protocols for ejaculated and epididymal ram sperm.     

Use of antioxidants reduce lipid peroxidation and improve quality of crossbred ram sperm during its cryopreservation

Ram sperm are subjected to extreme oxidative stress during their preservation at −196 °C resulting in reduced quality at post thaw. Therefore, the main objective of this study was to evaluate the effect of antioxidants taurine, quercetin and reduced glutathione on the post thaw quality of crossbred ram sperm. A total of twenty four ejaculates from six crossbred rams were collected and extended with tris-based extender with no antioxidant (Control), with taurine (40 mM), quercetin (5 μg/ml) and reduced glutathione (5 mM). The post thaw sperm quality was determined by percent sperm motility, live sperm count, intact acrosome and hypo-osmotic swelling test (HOST) reacted spermatozoa and lipid peroxidation was measured in terms of malondialdehyde (MDA) level both in seminal plasma and sperm cell. At post thaw, percent sperm motility and live sperm count were significantly (p < 0.05) higher for taurine than control and reduced glutathione but did not differ significantly (p > 0.05) from quercetin. The percent HOST reacted spermatozoa were significantly higher for taurine than control, quercetin and reduced glutathione. Seminal plasma MDA level was significantly (p < 0.05) lower for taurine than control and non-significantly lower than quercetin and reduced glutathione. However, spermatic MDA level did not differ significantly (p > 0.05) among the control and antioxidants. In conclusion, taurine at 40 mM reduced lipid peroxidation and improved post thaw sperm quality of cryopreserved crossbred ram semen. Further, transportation time of semen samples in an ice chest at 4–5 °C may be included as a part of equilibration period, when collection shed and frozen semen unit are located at a distance.     

Different concentrations of cysteamine and ergothioneine improve microscopic and oxidative parameters in ram semen frozen with a soybean lecithin extender

The aim of this study was to evaluate the effects of ergothioneine and cysteamine as antioxidant supplements in a soybean lecithin extender for freezing ram semen. Twenty-four ejaculates were collected from four rams and diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and cysteamine or ergothioneine (2, 4, 6 or 8 mM). Motility by CASA, viability, plasma membrane functionality (HOS test), total abnormality, lipid peroxidation, glutathione peroxidase (GPx) activity and capacitation status (CTC staining) were assessed after thawing. Using 6 mM of either antioxidant improved total motility. Cysteamine at 6 mM and ergothioneine at 4 and 6 mM improved viability and reduced lipid peroxidation (malondialdehyde concentration). Both antioxidants improved membrane functionality significantly, except at 8 mM. Progressive motility, kinematic parameters, GPx activity, capacitation status and sperm abnormalities were not influenced by the antioxidant supplements. In conclusion, cysteamine at 6 mM and ergothioneine at 4 or 6 mM seem to improve the post-thawing quality of ram semen cryopreserved in a soybean lecithin extender.     

Effect of lecithin nanoliposome or soybean lecithin supplemented by pomegranate extract on post-thaw flow cytometric,microscopic and oxidative parameters in ram semen

This investigation was carried out to study the effect of soybean lecithin 1.5% (wt/vol) (0, 2.5, 5 and 7.5 mg l⁻¹ pomegranate extract (PE)) or PE-loaded lecithin nanoliposome (0, 2.5, 5 and 7.5 mg l⁻¹) to Tris-based extender. Sperm motility (CASA), viability, membrane integrity (HOS test), abnormalities, mitochondrial activity, apoptosis status, lipid peroxidation, total antioxidant capacity (TAC)) and antioxidant activities (GPX, SOD) were investigated following freeze-thawing. No significant differences were detected in motility parameters, viability, membrane integrity, and mitochondria activity after thawing sperm between soybean lecithin and lecithin nanoliposomes. It was shown that PE5 significantly improved sperm total and progressive motility, membrane integrity, viability, mitochondria activity, TAC and reduced lipid peroxidation (malondialdehyde concentration). Moreover, the percentage of apoptotic sperm in PE5 extenders was significantly the lowest among other treatments. Sperm abnormalities, SOD and GPX were not affected by the antioxidant supplements. For apoptotic status, no differences were observed between soybean lecithin and lecithin nanoliposome. We showed that lecithin nanoliposome extender can be a beneficial alternative extender to protect ram sperm during cryopreservation without any adverse effects. It was also observed that regarding pomegranate concentration, PE5 can improve the quality of ram semen after thawing.     

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.     

Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility.     

Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters,lipid peroxidation and anti-oxidant activities

Ram semen contains sufficient quantities of superoxide dismutase (SOD) and much lower concentrations of glutathione peroxidase (GSH-PX) and catalase (CAT) to prevent oxidative damage. The anti-oxidant capacity of the sperm cell is limited, due to a small cytoplasmic component, which contains these anti-oxidants to scavenge the oxidants. However, the concentration of these anti-oxidants may decrease considerably by the dilution of the semen. The aim of the present work was to study the effect of two anti-oxidants, namely, glutamine and an amino acid solution (BME) in a Tris-based extender on ram sperm parameters, lipid peroxidation and anti-oxidant capacity after the cryopreservation/thawing process. Ejaculates collected from 4 Akkaraman rams were evaluated and pooled at 37 °C. Semen samples which were diluted with the tris-based extender containing glutamine (2.5 or 5 mM), BME (13 or 26%), and no anti-oxidants (control) were cooled to 5 °C and frozen in 0.25-ml French straws and stored in liquid nitrogen. Frozen straws were thawed individually at 37 °C for 20 s in a water bath for evaluation. The freezing extender supplemented with 5 mM glutamine led to higher motility rate (68.0 ± 4.4%) and hypo-osmotic swelling test (HOST) (64.1 ± 5.5%), when compared to glutamine (2.5 mM) and BME (13 and 26%) (P < 0.05). No significant differences were observed regarding sperm motility and HOST, following the supplementation of the freezing extender with glutamine 2.5 mM and BME (13 and 26%) after thawing. CAT activity remained significantly higher following the addition of glutamine 5 mM (6.4 ± 0.9 kU/g protein), compared to the other treatments (P < 0.01). The anti-oxidants at different levels were not effective in the elimination of malondialdehyde (MDA) formation and maintenance of SOD activities, when compared to the control (P < 0.05). Findings showed that glutamine (5 mM) supplementation in semen extenders, was of greater benefit to frozen–thawed ram sperm. Future efforts are needed to find the appropriate anti-oxidants and their effective concentrations to improve post-thaw sperm parameters (e.g. motility, membrane integrity, fertility) and anti-oxidant activities when frozen–thawed ram sperm is used.     

Differential staining for live and dead sperm of honey bees

ABSTRACT Seven staining techniques were modified and tested for differentially staining the live and the dead sperm cells for the honey bee (Apis mellifera L.). The eosin Y staining method was found to be a simple technique by which the live cells stain bluish purple whereas the dead cells stain bright yellow to greenish yellow. Therefore, it produces a strong contrast between the dead and live sperm cells, and appears to be the most suitable supravital staining method for evaluating the viability of honey bee sperm cells. The significance of supravital staining techniques in assessing the quality of sperm cells during cryopreserving sperm cells is discussed.     

Automated analysis of rabbit sperm motility and the effect of chemicals on sperm motion parameters.

Appropriate software settings and optimum procedures were determined for the measurement of the motion parameters of rabbit spermatozoa by the CellSoft (Cryo Resources Ltd., Montgomery, NY) computer-assisted digital image analysis system. The system was used to follow motion parameter changes occurring in spermatozoa incubated for 6 hr with or without exposure to chemicals. Mean amplitude of lateral head displacement (AALH) increased over the 6 hr period, while curvilinear velocity (Vc) first increased and then decreased. Values for linearity (Lin), or beat cross frequency (BCF), were unchanged. The majority of spermatozoa progressed linearly, with rapid rotation of the sperm head, but subpopulations of spermatozoa with different swimming patterns appeared after 1-3 hr of incubation. Percentage motile sperm and Vc were most sensitive to the action of the compounds (pyrogallol, hydroquinone, ammonium oxalate, triethyl phosphite, and pinocolyl alcohol), while BCF was least affected. The decline in percentage of motile sperm was dependent on duration of exposure and chemical concentration. Mean Vc of the sperm population decreased rapidly upon chemical exposure and remained at a low value until motility ceased. The initial decrease in Vc was dependent on the concentration of the added compound. Motion-based indices--motility concentration (MCI50), motility time (MTI50), and velocity (VI)--were defined and used as toxicological endpoints. The rank order of these indices, the end point of the neutral red in vitro assay for cytotoxicity, and LD50 values for the five compounds were the same, suggesting that chemical inhibition of sperm motility may be useful as a method for the in vitro assessment of chemical cytotoxicity.     

The relationship between ram sperm head morphometry and fertility depends on the procedures of acquisition and analysis used

Sperm head morphometry is a parameter in the evaluation of semen that has been associated with fertility in two ways: comparing morphometric measures between predefined groups of fertility; or analyzing morphometric data by multivariate techniques to identify cell populations. We analyzed the morphometry of ram sperm head by three procedures and checked its relationship with male fertility. A Computer-Aided Sperm Morphometric Assessment procedure (CASMA), an image analysis software (NIS-Elements) in combination with an optical microscope (MO-NIS) and this image analysis software in combination with a scanning electron microscope (SEM-NIS) were used. Eight morphometric parameters were assessed: length, width, area, perimeter, ellipticity, form factor, elongation and regularity. We observed significant differences between the morphometric data of sperm head obtained with three study procedures. The CASMA procedure shows the highest values for all parameters and the SEM-NIS procedure the lowest. The analysis of a semen sample, when only the mean of morphometric parameters is used to describe the cell population, is too limited to interpret their fertilizing capacity. It is essential to analyze the complex structure of the samples by defining subpopulations by multivariate methods. With few exceptions, the means of each morphometric parameter differ between the three subpopulations analyzed in each procedure. Only the subpopulations obtained with the MO-NIS procedure showed a significant correlation with male fertility. In short, it is necessary to establish an instrumental standard for the analysis of sperm morphometry to obtain reliable results and we believe that the MO-NIS system presents these basic requirements.     

Morphometry of porcine spermatozoa and its functional significance in relation with the motility parameters in fresh semen

Both the study and the relationship between sperm design and sperm function have been a target of several researchers. In our study we have evaluated the relationship between the morphometry of sperm head and midpiece as well as the relationship between morphometry of these two spermatic components and sperm motion characteristics in the boar. Analysis of regression (lineal and multiple) and principal components analysis were used for the study of these relationships. Semen samples from five Iberian boars were taken for analysis. Analysis of morphometry was assessed by CASMA system and motility by CASA system. Sperm midpiece showed a significant relationship (positive or negative, depending on the morphometric parameter evaluated) with sperm head. VSL, LIN, STR, BCF and VAP showed a significant relationship with several head and midpiece morphometric parameters. Finally, through the analysis of multiple lineal regression we obtained several statistical models that predict STR, LIN, VCL, ALH, BCF, PC1 and PC2 (the last two variables have been obtained from a principal components analysis) as a function of one, two or three morphometric parameters. Our results suggest a co-evolution of sperm head and midpiece and in addition that sperm motion characteristics of porcine spermatozoa are influenced by morphometry of head and midpiece.     

Seasonality of LH,testosterone and sperm parameters in spider monkey males (Ateles geoffroyi)

There are no reported data on hormonal fluctuations in black‐handed spider monkey males. On previous research about the reproductive physiology of this monkey we have found that during the dry season females show ovulatory estrogen peaks and males present the best quality semen. As part of an ongoing research, in this study we assessed seasonal variations in the concentration of serum luteinizing hormone (LH) and testosterone (T) in three adult spider monkey males to corroborate the seasonal reproductive synchrony. At the same time sperm count and motility were evaluated to search for any correlation between those sperm parameters and hormonal concentrations. We took blood and semen samples (by electroejaculation) of anesthetized males throughout the rainy (June–September) and dry (October–May) months. Our results revealed that T and LH were higher throughout the dry season and there was a significant correlation between T concentration and sperm count. Although higher during the dry season, sperm motility tended to correlate with testosterone and LH levels. These results demonstrated that black‐handed spider monkeys have a tendency to show a seasonal pattern of reproduction being the dry season the most likely time to achieve fertilization. Am. J. Primatol. 71:427–431, 2009. © 2009 Wiley‐Liss, Inc.     

Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm

Assisted reproduction using frozen-thawed semen has practical advantages, although cryopreservation is detrimental to sperm fertility in most mammals. We examined the influence of cryopreservation and reactive oxygen species (ROS) on ram sperm DNA stability (using SCSA), lipid peroxidation (LPO), chlortetracycline fluorescence (CTC) patterns, motility and viability. In Experiment 1, DNA integrity, LPO, CTC, motility and viability tests were performed on fresh and cryopreserved sperm after 0, 6, and 24 hr in synthetic oviductal fluid (SOF). In Experiment 2, fresh sperm were incubated in serum-free SOF (SOF-S; 1, 4, and 24 hr) with 0, 50, 150, or 300 microM H2O2 then assayed. Cryopreservation increased the percentage of sperm with a high DNA fragmentation index (%DFI), decreased the percentages of motile and viable sperm at thawing (0 hr), but did not affect LPO. H2O2 (150 or 300 microM) increased %DFI after 24 hr. LPO or sperm viability were not affected by H2O2, although most motility parameters decreased. H2O2 decreased the percentage of chlortetracycline pattern F sperm at 4 hr and increased the percentage of acrosome-reacted sperm (pattern AR) after 1 hr. Pooled data of Experiment 2 showed LPO was positively correlated with SCSA (r = 0.29 to r = 0.59; P < 0.05 to P < 0.01), while most motility parameters and the percentage of viable sperm were negatively correlated with LPO (r = -0.30 to r = -0.38; P < 0.05 to P < 0.01). LPO was positively correlated with the percentage of pattern AR sperm (r = 0.33; P < 0.01). Cryopreservation and H2O2 promote DNA instability in ram sperm, though motility is a more sensitive indicator of oxidative stress than the other parameters investigated.     

Survival at 20° C and cryopreservation of isolated sperm cells from Zea mays pollen grains

Summary The survival of isolated sperm cells from maize pollen grains at 20° C and their cryopreservation were studied by means of the fluorochromatic reaction (FCR) test. An osmotic pressure of 500 mOsmol, a pH of 7 adjusted with MOPS buffer, and the ten-fold dilution of Brewbacker and Kwack salts enabled isolated sperm cells to survive for a maximum of 35 h at 20° C. The addition of 3 mM of calcium chloride or 0.1 % glucose also slightly improved survival. The addition of 1% glutamine gave the best percentage of FCR-positive cells at 20 h. The addition of all of these substances in a unique medium provoked the formation of spermcell aggregates that were FCR positive. With respect to cryopreservaton, 70% of the isolated sperm cells remained FCR positive after freezing at -80° C and quick thawing at 37° C in the selected survival medium.