The influence of age and breed on stallion semen

This study reports on the variation in semen quality and in spermatozoal and behavioral characteristics of 168 stallions representing 9 breeds and ranging in age from 2 to 26 yr. Semen samples were collected into an artificial vagina and the number of mounts and urethral pulsations per semen sample were recorded. Semen characteristics were examined for total volume, gel-free volume, gel volume, color score, mass activity, nonmotile spermatozoa, dead spermatozoa, semen density, spermatozoa concentration, total number of spermatozoa and semen pH. Morphological characteristics of the spermatozoa included abnormal heads, abnormal mid-pieces, abaxial mid-pieces, protoplasmic droplets and abnormal tails. Sources of variation were evaluated and the overall means calculated by least-squares analyses of variance for nonorthogonal data. The significance of breed effects and between stallion variability were estimated using mixed-model procedures. All semen characteristics with the exception of color and urethral pulsations had significant variation due to age. Semen quality (gel-free volume, sperm concentration, total sperm numbers and sperm abnormalities) was poorest in stallions under 3 yr of age and over 11 yr. Significant breed variation was apparent in most characteristics except for pH, semen color, abnormal midpieces and urethral pulsations. It is recommended that both the age and breed of stallion be taken into consideration when evaluating stallion semen.     

Is there a relationship between sperm chromosome abnormalities and sperm morphology?

This review explores the relationship between sperm chromosomal constitution and morphology. With the advent of techniques for obtaining information on the chromosome complements of spermatozoa, this relationship has been studied in fertile men and in men with a high frequency of chromosomal abnormalities. Using human sperm karyotype analysis, no relationship between sperm chromosome abnormalities and morphology was found in fertile men, translocation carriers or post-radiotherapy cancer patients. Fluorescence in situ hybridization (FISH) analysis has not generally revealed a specific association between morphologically abnormal sperm and sperm chromosome abnormalities, but has indicated that teratozoospermia, like other forms of abnormal semen profiles (aesthenozoospermia, oligozoospermia) is associated with a modest increase in the frequency of sperm chromosome abnormalities. However, FISH studies on some infertile men and mouse strains have suggested that certain types of morphologically abnormal spermatozoa, such as macrocephalic multitailed spermatozoa, are associated with a very significantly increased frequency of aneuploidy. Thus, there may be an association between sperm morphology and aneuploidy in infertile men with specific abnormalities.     

Collection of semen and artificial insemination of alpacas

Semen collection and artificial insemination have not yet been fully developed in the alpaca. Thus, we collected semen from 7 males using a modified artificial vagina placed inside a dummy. Forty adult female alpacas, previously induced to ovulate with hCG, were artificially inseminated with fresh undiluted semen by laparoscopy or by cervix. The Chisquare test was used to determine differences in the fertility rate of the 2 insemination methods. The mean duration of copulation, semen volume, sperm concentration and the percentages of live spermatozoa and normal spermatozoa were 21.6 min, 1.9 ml. 147,500/mm(3), 69.6% and 75.9%, respectively. There were 6.7% abnormal heads, 12.3% abnormal tails and 3.8% cytoplasmic droplets. The consistency of semen was viscous and formed a coagulum. The pH was 7.2, and the semen was milky white in color. The duration of copulation was comparable to natural copulation, and semen characteristics reflected those of the natural ejaculate. The percentage of pregnancy was 68%, with no differences due to method of semen deposition (laparoscopy, 67%; cervix, 73%).     

Morphometry of normal and teratozoospermic canine sperm heads using an image analyzer: work in progress

Combining the traditional morphologic evaluation of spermatozoa with computer assisted image analysis adds randomness, objectivity, repeatability and accuracy to morphometric measurements. We collected semen from 10 fertile, normospermic dogs aged 1 to 7 yr and from 3 teratozoospermic breed-matched dogs. Sperm head morphology was examined in Giemsa-stained smears by light microscopy, using a computer-assisted image analyzer and by transmission electron microscopy. We found significant variation in sperm head area, length, width and degree of roundness among normospermic individual dogs, indicating that it would be necessary to examine many more dogs before the size and shape of normal dog spermatozoa could be determined. The normospermic dogs were used as controls for the teratozoospermic cases. Case 1: A 2-yr-old subfertile Cavalier King Charles Spaniel had semen with small and narrow-based sperm heads and a proximal cytoplasmic droplet in most of the spermatozoa. With the image analysis system, sperm heads were shown to be smaller and more oval than in normospermic dogs. The variatons in size and shape were similar in magnitude to those of control dogs. An examined infertile half-brother had similar semen quality. Case 2: A 3-yr-old Petit Basset Griffon Vendeen with 2 unsuccesfull matings exhibited spermatozoa with severe abnormalities. Measured by image analyzer, sperm heads were irregular in shape and very small in area. One of the two littermates examined had semen of the same quality as the case dog. Case 3: A 3-yr-old fertile Golden Retriever had semen with giant sperm heads in about 50% of spermatozoa. Image analyzing results revealed 2 populations of different sized sperm heads. Giant heads consisted of 52.2% of all spermatozoa. The results of the study reported here suggest that the image analysis technique may be useful in evaluating structural changes in sperm morphology, supplementing visual assessment that is used in conventional methods.     

Relationship between heparin binding characteristics and ability of human spermatozoa to penetrate hamster ova

Heparin binding site affinity and density on human spermatozoa were compared between fertile and infertile men with normal or abnormal results in the zona-free hamster ova-sperm penetration assay (SPA). A portion of fresh semen from fertile donors and potentially infertile men was processed through the SPA while the remainder of the ejaculate was used to quantitate heparin binding on spermatozoa. Saturation binding assays with [3H]heparin (15-375 nM) were analysed for 3 groups of men: (1) infertile patients with abnormal SPA results, (2) infertile patients with normal SPA results and (3) fertile donors. The heparin binding site density was significantly higher in men who possessed normal SPA results (infertile men and fertile donors) than in infertile men with abnormal scores in the SPA. There was no difference in heparin binding affinity between the three groups. These findings suggest that the heparin binding-site density may be related to the ability of human spermatozoa to undergo successfully the acrosome reaction.     

Testicular degeneration in a male goat. A case report

Semen samples from an 18-month-old infertile Lamancha buck contained less than 10% motile spermatozoa and a high incidence of morphological abnormalities. Degenerate detached heads, tightly coiled tails, and thickened midpieces were the prominent morphological defects, affecting 46%, 30%, and 15% of ejaculated spermatozoa, respectively. At necropsy, the diagnosis of testicular degeneration was made, characterized by focal, noninflammatory seminiferous tubule occlusion and mineralization, with a more generalized sloughing of tubular germinal epithelium. When examining spermatozoa morphology from various sites of the excurrent ducts, detached heads were readily seen in efferent duct fluid (> 30% incidence) and the coiled tails and other midpiece defects became prominent with passage through the caput epididymis.     

Reversible meiotic abnormalities in azoospermic men with bilateral varicocele after microsurgical correction.

Because of a possible relationship between microenvironmental disturbances and meiotic abnormalities and of a straight relationship between lower-quality semen in patient carrying a varicocele and first meiotic non-disjunction, bilateral bipolar testicular biopsies are realized according the thermic differential gradient described in varicocele. Systematic meiotic studies of multiple testicular biopsies from 65 azoospermic men with bilateral varicocele were done in a multi-centric study on microsurgical correction of bilateral varicocele with microthermic intra-operative evaluation using minimally invasive thermal microsensors (Betatherm 10K3MCD2). In the present study abnormal temperature raising, histomorphometric abnormalities (spermatocyte arrest) and meiotic abnormalities (class IIC) are strongly correlated. In the ten patients submitted to another testicular biopsy procedure six months after surgery for TESE, normal thermal differential is registered and no meiotic abnormalities recurrences are found.     

Seminal collection, seminal characteristics and pattern of ejaculation in llamas

Semen was collected from 10/10 llamas during 26/30 (87%) collection attempts using an artificial vagina mounted inside a surrogate female. For the 26 semen collections, the duration of copulation (mount to dismount) with the artificial vagina was 31.7 +/- 12.0 min (mean +/- SD). Seminal pH was 8.1 +/- 1.1, and seminal volume per collection was 3.0 +/- 1.9 ml. Sperm concentration per collection was 1.0 +/- 0.8 x 10(6) sperm/ml, total number of spermatozoa was 2.9 +/- 3.1 x 10(6), total sperm motility was 23.7 +/- 20.0%, and the percentage of morphologically normal spermatozoa was 39.7 +/- 18.5%. Morphologically abnormal spermatozoa were categorized according to abnormal heads (20.1 +/- 19.9%), tail-less heads (8.7 +/- 8.9%), abnormal acrosomes (12.9 +/- 12.4%), abnormal midpieces (1.0 +/- 3.7%), cytoplasmic droplets (11.1 +/- 12.4%), and abnormal tails (6.6 +/- 12.0%). There were 0.3 +/- 0.3 million motile, morphologically normal spermatozoa per collection: less than 1000 during the first 5 min of copulation, 0.01 +/- 0.01 x 10(6) between 5 and 10 min of copulation, 0.04 +/- 0.08 x 10(6) between 10 and 15 min of copulation, 0.09 +/- 0.21 x 10(6) between 15 and 20 min of copulation, and 0.15 +/- 0.28 x 10(6) between 20 min and the end of copulation.     

Sperm morphological abnormalities appearing in the male rabbit reproductive tract

The role of the excurrent duct system in producing and/or eliminating morphologically abnormal spermatozoa may modify the semen parameters and interfere with sperm fertilizing capacity. To study this process, changes in the morphology of spermatozoa during their transit through the reproductive tract in sexually mature rabbits were investigated. The incidence of head, midpiece and tail abnormalities as well as of multiple defects in a single spermatozoon, and the position of the cytoplasmic droplet along the sperm midpiece were evaluated in samples from the testis, 6 regions of the epididymis and the vas deferens. Spermatozoa were characterized by rapid migration of the cytoplasmic droplet when passing from the proximal to the distal caput of the epididymis, and spermatozoa with no droplet predominated in the distal epididymis and vas deferens. In passing from the testis to the proximal caput of the epididymis, the incidence of spermatozoa with an abnormal midpiece and those with multiple defects decreased significantly. The proportion of spermatozoa with abnormal heads was also lower in the testis, but no statistically significant differences were found, whereas there was no change in the proportion of those with abnormal tails. These results indicate that there must be a mechanism for the disposal of defective spermatozoa. No evidence of spermiophagy by luminal macrophages was observed in the extracts, although a few spermatozoa exhibited signs of degeneration, suggesting, that although intraepithelial phagocytosis has not been clearly demonstrated in the nonexperimental rabbit, sperm cells may undergo a form of autolysis within the lumen of the duct.     

Human spermatozoa with large heads and multiple flagella: a quantitative ultrastructural study of 6 cases

Macrocephalic spermatozoa of six men were studied. In all cases, sperm concentration, proportion of live spermatozoa and sperm motility were very low. A range of ultrastructural abnormalities was found, essentially comprising a threefold increase in nuclear volume and acrosomal hyperdevelopment and malformation. There were on average 3.6 flagella for each sperm head found in the semen, some tails were separate from heads. The various defects appeared with great constancy in all of the six cases: this homogeneity indicated the existence of a defined semen profile whose most significant expression was sterility. In four of the cases large incidences of different flagellar abnormalities were also noted; whether these flagellar abnormalities are intrinsic to the above profile is not clear. Although the increase in nuclear volume suggests a disturbance in meiosis, its association with defective nuclear elongation would also indicate the existence of one or more anomalies of spermiogenesis. These results were discussed in relation to abnormalities already reported in other species either spontaneously in cases of mutations, or by experimental inhibition of microtubular structures.     

Human spermatozoa with large heads and multiple flagella: a quantitative ultrastructural study of 6 cases

Macrocephalic spermatozoa of six men were studied. In all cases, sperm concentration, proportion of live spermatozoa and sperm motility were very low. A range of ultrastructural abnormalities was found, essentially comprising a threefold increase in nuclear volume and acrosomal hyperdevelopment and malformation. There were on average 3.6 flagella for each sperm head found in the semen, some tails were separate from heads. The various defects appeared with great constancy in all of the six cases: this homogeneity indicated the existence of a defined semen profile whose most significant expression was sterility. In four of the cases large incidences of different flagellar abnormalities were also noted; whether these flagellar abnormalities are intrinsic to the above profile is not clear. Although the increase in nuclear volume suggests a disturbance in meiosis, its association with defective nuclear elongation would also indicate the existence of one or more anomalies of spermiogenesis. These results were discussed in relation to abnormalities already reported in other species either spontaneously in cases of mutations, or by experimental inhibition of microtubular structures.     

Attempts on freezing the Greylag (Anser anser L.) gander semen

Semen of Greylag (Anser anser L.) ganders was frozen according to a method previously elaborated by the authors for freezing the White Koluda gander semen. Semen was collected from five to eight Greylag ganders, twice a week during three succeeding reproductive cycles, by dorso-abdominal massage. Semen samples were diluted in the ratio of 1:1 or 2:1 (two parts semen: one part diluent) with EK diluent, supplemented by 6% DMF, equilibrated and pre-frozen to -140 degrees C at a rate 60 degrees C/min, before being transferred into liquid nitrogen container. Semen samples thawed in a water bath of 60 degrees C were used for twice a week insemination in a volume of 200 microl. Three Greylag and three White Koluda geese were involved in frozen-thawed semen fertilizing ability test. The reproductive cycle of wild geese lasts usually about 6-7 weeks. The ejaculate volume (30-140 microl) and sperm concentration (10x10(6) to 150x10(6) ml(-1)) are much lower than these of domestic ganders, but spermatozoa morphology is similar, particularly while compared to 1-year-old White Koluda ganders semen. There are about 90% of live spermatozoa and about 30% of live morphologically normal cells in Greylag gander fresh semen. The Greylag gander spermatozoa susceptibility to cryopreservation procedure is as high as in domestic ganders. Dilution ratio 2:1 resulted in higher number of live spermatozoa, which withstood cryoinjury stress. In relation to fresh semen about 60% of spermatozoa remained intact (on the basis of light microscope examination) in the frozen-thawed semen. Insemination of frozen-thawed semen resulted in 37.5% of fertile eggs in Greylag and 25.0% in White Koluda geese. Low fertility rate was caused by an insufficient number of live normal spermatozoa used for insemination (about three million in every dose).     

Nuclear maturity and morphology of human spermatozoa selected by Percoll density gradient centrifugation or swim-up procedure

The selection of motile human spermatozoa, from fertile and infertile semen samples was compared by using Percoll density gradient centrifugation or the swim-up procedure. Selected spermatozoa were evaluated according to their motility, % normal forms, nuclear maturity (aniline blue staining, acridine orange staining, ethidium bromide uptake and SDS nuclear decondensation). These methods showed differences between fertile and infertile men. The swim-up procedure, based on motility, resulted in greater proportions of motile spermatozoa and eliminated mainly tail abnormalities. Percoll gradient separation, based on density, selected oval-headed spermatozoa with good motility. Nuclear maturity level was improved by both methods but Percoll gradient separation generally resulted in spermatozoa with better nuclear maturity than those selected by the swim-up procedure.     

New data on aberrant spermatozoa in the ejaculate of Sus domesticus

This paper describes 16 new types of aberrant spermatozoa observed by scanning electron microscopy in the ejaculate of two healthy, sexually mature Landrace boars. The new anomalies observed were 1) spermatozoa with folded tail and abnormal head; 2) tailless spermatozoa with an abnormal connecting piece; 3) immature spermatozoa with two tails of the same length, fused and coiled; 4) spermatozoa with two tails of the same length, fused and coiled, and a small, rounded head; 5) spermatozoa with two fused tails and a wide head; 6) spermatozoa with three tails of the same length, fused and coiled; 7) immature spermatozoa with two heads and two fused tails; 8) spermatozoa with two heads, one at each tip of the tail; 9) spermatozoa with a short, folded tail and a triangular head; 10) spermatozoa with a short tail lacking the intermediate piece; 11) spermatozoa with a short tail, without the main piece and with a long intermediate piece; 12) spermatozoa with a short tail, without the main piece and with a rough head; 13) spermatozoa with small, rounded head; 14) spermatozoa with small, aberrant heads; 15) spermatozoa with small, bacillary heads; and 16) immature spermatozoa with tapering heads.     

Examen ultrastructural quantitatif des spermatozoides humains: Intérêt,méthode,valeurs de référence,indications

The use of transmission electron microscopy in clinical andrology is complicated by problems of classification and interpretation related to the extraordinary pleomorphism of human spermatozoa, even in fertile men. Based upon our experience of more than 650 cases, this paper describes the morphological abnormalities seen and proposes a simplified classification which is the basis of our approach to the quantitative ultrastructural assessment of human spermatozoa. The mean and maximum incidences of the various abnormalities are provided for 37 fertile ejaculates. These reference values, particularly the threshold value of 50% abnormal acrosomes, constitute our interpretation criteria. Finally, the indications for this type of extended evaluation are defined as unexplained (idiopathic) infertility, severe asthenozoospermia, and teratozoospermia characterized by a high level of abnormal heads.     

The occurrence of reactive oxygen species in the semen of males from infertile couples

The aim of this pilot study was to establish a reactive oxygen species (ROS) evaluation method as a step in the routine diagnosis of men from infertile couples, which attend the Centre of Assisted Reproduction at the Teaching Hospital in Olomouc. Standard semen analyses were performed manually according to WHO guidelines. The number of peroxidase-positive leukocytes in the semen was determined using the Endtz test. The levels of ROS were estimated by chemiluminescence assay using luminol (5-amino-2,3 dihydro-1,4 phthalazinedione) as a probe. The semen samples were collected from 68 patients. Normospermia was found in 15 patients (22.1 %). The semen samples of 3 normospermic patients were classified as ROS-positive. Elevated ROS production was recorded in all subgroups of patients irrespective of any pathology found. We confirmed that spermatozoa might be the source of ROS as well as the seminal leukocytes. Apart from the leukocytes, sperm cells with residual cytoplasm and immature spermatozoa are considered to be a major source of ROS. Thus it is suggested that sperm morphology abnormalities should be evaluated more carefully.     

Seasonal variations of semen quality in male goats: study of sperm abnormalities

The type, frequency and seasonal variation of sperm abnormalities in male goats inhabiting in Mediterranean region were evaluated. Semen was collected twice a week with an artificial vagina from 9 10-month-old Murciano-Granadina male goats weighing 41 to 48 kg. Distal cytoplasmic droplets and acrosomal damage were the predominant abnormalities. Increase in age (10 to 27 months) did not reveal itself as a main effect on the occurrence of sperm abnormalities. All types of sperm abnormalities studied showed significant (P<0.01) seasonal variation. The poorest quality semen was collected during winter and spring. In spite of a seasonal variation in sperm quality, semen of Murciano-Granadina goats in the Mediterranean area is good all year long, since the proportion of abnormal spermatozoa was well within the accepted range for normal fertility.     

Expression of a2 Vacuolar ATPase in Spermatozoa is Associated with Semen Quality and Chemokine-Cytokine Profiles in Infertile Men

Background

A number of laboratory tests have been developed to determine properties of spermatozoa quality but few have been adopted into routine clinical use in place of the WHO semen analysis. We investigated whether Atp6v0a2 (a2 isoform of vacuolar ATPase) is associated with abnormal semen quality and changes in chemokine-cytokine profiles in infertile men.

Patients and Methods

Semen samples were collected from 35 healthy donors and 35 infertile men at the Andrology laboratory from August 2011 to June 2012. The levels of Atp6v0a2 mRNA and protein, and its localization in spermatozoa were determined. a2NTD (the N-terminal portion of Atp6v0a2) and secreted chemokine-cytokine profiles in seminal fluid were measured.

Results

Atp6v0a2 protein (P<0.05) and mRNA (P<0.05) in spermatozoa from infertile men were significantly lower than those from fertile men. Fluorescent microscopy revealed that Atp6v0a2 is mainly expressed in the acrosomal region. Infertile men’s seminal fluid had significantly lower G-CSF (P<0.01), GM-CSF (P<0.01), MCP-1 (P<0.05), MIP-1α (P<0.01) and TGF-β1 (P<0.01) levels when compared to the seminal fluid from fertile men. Seminal fluid a2NTD levels were significantly correlated with G-CSF (P<0.01), GM-CSF (P<0.01), MCP-1 (P<0.05), MIP-1α (P<0.01) and TGF-β1 (P<0.01) which are key molecules during the onset of pregnancy.

Conclusion

These results suggested that a critical level of Atp6v0a2 is required for the fertile spermatozoa and its decreased level in spermatozoa could be used to predict male infertility. This study provides a possibility that Atp6v0a2 could be potentially used as a diagnostic marker for the evaluation of male infertility.     

Decreased protein tyrosine phosphorylation and membrane fluidity in spermatozoa from infertile men with varicocele

Varicocele is a prevalent pathology among infertile men. The mechanisms linking this condition to infertility, however, are poorly understood. Our previous work showed a relationship between sperm functional quality and the ability of spermatozoa to respond to capacitating conditions with increased membrane fluidity and protein tyrosine phosphorylation. Given the reported association between varicocele, oxidative stress, and sperm dysfunction, we hypothesized that spermatozoa from infertile patients with varicocele might have a combined defect at the level of membrane fluidity and protein tyrosine phosphorylation. Semen samples from infertile patients with and without grade II/III left varicocele were evaluated for motion parameters (computer-assisted semen analysis [CASA]), hyperactivation (CASA), incidence and intensity of protein tyrosine phosphorylation (phosphotyrosine immunofluorescence and western blotting), and membrane fluidity (Laurdan fluorometry), before and after a capacitating incubation (6 hr at 37 degrees C in Ham's F10/BSA, 5% CO(2)). Spermatozoa from varicocele samples presented a decreased response to the capacitating challenge, showing significantly lower motility, hyperactivation, incidence and intensity of tyrosine phosphorylation, and membrane fluidity. The findings reported in this article indicate that the sperm dysfunction associated to infertile varicocele coexists with decreased sperm plasma membrane fluidity and tyrosine phosphorylation. These deficiencies represent potential new pathophysiological mechanisms underlying varicocele-related infertility.     

Selenium,Copper and Zinc in Seminal Plasma of Men with Varicocele,Relationship with Seminal Parameters

Varicocele has been associated with decrease in seminal parameters. Selenium (Se), copper (Cu), and zinc (Zn) are trace elements essential for normal spermatogenesis of mammals and play a critical role as antioxidant defense system enzymes. Se, Cu, and Zn are associated with sperm quality in fertile and infertile men. However, there is little information about Se, Cu, and Zn concentrations in semen in patients with varicocele and its association with seminal parameters. The purpose of this study was to determine the concentrations of Se, Cu, and Zn in semen of patients with varicocele and the relationship with seminal parameters. Total Reflection X-Ray Fluorescence was used for the fist time in the seminal fluid analysis. The concentration of selenium in men with varicocele was smaller than the normozoospermic group, while no differences were observed for both concentrations of zinc and copper. A significant positive correlation between zinc and selenium concentration was observed. Selenium in seminal plasma correlates with a good spermatozoa concentrations, motility, and morphology. Additionally, a significant positive correlation was observed between zinc levels and sperm count. In conclusion, a decrease in selenium concentration was associated with detriment of seminal parameters. A study should be conducted to evaluate the benefits of both zinc and selenium supplementation to improve seminal parameters in patients with varicocele.