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Retained cytoplasmic droplets (CD) provide the most abundant sperm abnormality in boar and reduce fertility. It is still unclear as to whether high CD rates in semen portions are compensable. The aim was to explore the impact of CD in relation to quantitative and qualitative sperm traits on fertility performance of sows. Retrospective data analysis of 1497 inseminations was performed. Ejaculates (n = 260) were assigned to three groups with low (

This work aimed to establish whether the temperature humidity index (THI) under different intertropical zones affects the retention of cytoplasmic droplets (CDs), sperm function and DNA integrity in boars. With this purpose, two separate studies were devised. In the first one, 49 boars from six farms were collected every 45 days (230 ejaculates). THI were measured daily, and sperm parameters were evaluated. Boars were classified into three groups based on the incidence of ejaculates having more than 25% spermatozoa with CDs: persistent (at least three consecutive ejaculates), moderate (less than three ejaculates) and absent (no ejaculate having ≥25% spermatozoa with CDs). Farms were classified based on THI through cluster analysis into two groups. In the second study, 32 liquid-stored semen samples were classified based on three cluster analysis: low and high incidence of proximal (PCDs), distal (DCDs) CDs and a combination of PCD and DCDs. high THI farms presented significantly (p < .05) higher proportions of boars with moderate and persistent incidence of CD than those with low THI. In study 2, the presence of PCDs was negatively correlated with sperm DNA integrity (r = -0.691; p < .01). However, differences between groups were more apparent when ejaculates were classified based on both PCDs and DCDs than when PCDs or DCDs were considered separately. In conclusion, classification of boars according to the severity and persistence of CDs in boars allows understanding more clearly the dynamics of CD retention and the effects of ambient temperature and relative humidity. Additionally, the joint analysis of both PCDs and DCDs is necessary in routine sperm quality analyses.

Morphological changes in the caput, corpus and cauda epididymides were studied in this experiment. The objective was to study morphological changes in goat spermatozoa using the Red Sokoto goat. Progressive motile spermatozoa were tint observed in the caput epididymis (5-7%) and was highest at the cauda epididymis (60%). Progressive motility for the corpus epididymis ranged between 20-30%. Cytoplasmic droplets were lost as the spermatozoa moved up the epididymis. Proximal cytoplasmic droplet was significantly (P

A seven week study was conducted to determine the effect of three dietary levels of cottonseed meal (0%, 15%,30%) on the occurrence of spermatozoa morphological abnormalities in mature Rhode Island White (RIW) breeder cocks. Abnormalities of the acrosome, sperm head, cytoplasmic droplets and mid piece and sperm tail were investigated in 180 ejaculates collected between October and November. All types of abnormalities were variable between treatments and between extent of feeding (weeks). The values (2.90,5.30 and 8.63) on sperm head abnormalities were significantly different (P

Objective: To evaluate normal spermatozoa morphometry and ultrastructure in estuarine crocodile (Crocodylus porosus). Methods: Four adult male crocodiles aged between 12-15 years, with an average snout to tail length of (3.15±0.01) m were selected for this study. Manipulation of the phallus digitally from the base of the penis was performed on four adult male crocodiles to facilitate the flow of semen to the sulcus. Semen was collected from all individuals for spermatozoa morphometry and ultrastructure study. Morphometry analysis was performed from eosin-nigrosin stained spermatozoa sample. Scanning electron microscopy was conducted to observe the surface ultrastructure of spermatozoa. Results: The morphology of crocodile spermatozoa was made up of acrosome, head, and tail which corresponded to (5.55±1.20) μm, (12.74±1.57) μm, and (70.67±4.40) μm, respectively. The total length of spermatozoa in estuarine crocodile was measured at (88.96±0.52) μm. The most common spermatozoa abnormalities found in the samples were cytoplasmic droplets. Conclusions: Normal morphology, morphometry of spermatozoa in estuarine crocodile which are vermiform shaped-head with a long tail along with questionably high cytoplasmic droplets count are described in this study.

The morphological characteristics of different sperm cells (normal, abnormal, and immature) in the peregrine falcon during the reproductive season were analysed. We also classified the main sperm defects found in semen. Semen samples were collected from mature peregrine falcons via cloacal massage and stained with Diff-Quik stain. The percentages of normal, abnormal, and immature sperm cells were determined by bright-field optical microscopy. The number of normal spermatozoa were greater at the initial stage and subsequently decreased during the middle and later stages of the reproductive season (p < 0.01). In contrast, the percentage of abnormal spermatozoa increased significantly in the middle and end stages of the reproductive season (p < 0.05), whereas the proportion of immature spermatozoa remained stable during the study. Head defects represented the greatest proportion of morphological abnormalities, followed by the defects in the tail and midpiece regions. A small percentage of multiple defects and cytoplasmic droplets were also observed in the falcon spermatozoa. The findings of this study might be important for the development of future conservation protocols for falcon sperm.

Retained cytoplasmic droplets (CD) are the most frequent sperm abnormality in boar semen. A high incidence of CD is associated with subfertility, but the underlaying reasons are not well understood. The storage of extended semen might augment the adverse effects of CD on essential steps towards fertilization, such as capacitation. The aim of this study was to examine whether the enhanced presence of CD in boar semen influences sperm’s response to the capacitation stimulus bicarbonate during long-term semen storage. Extended semen samples (n = 78) from 13 artificial insemination centers were analyzed using a flow cytometric calcium influx assay. Samples with &gt, 15% of CD showed a reduced specific response to bicarbonate and a higher non-specific destabilization after storage for 96 h and subsequent incubation at 38 °C in three variants of Tyrode`s medium (p &lt, 0.05). The size of the bicarbonate-responsive sperm population was inversely correlated with the presence of CD-bearing sperm (r = −0.61, p &lt, 0.01). Samples with ≤15% and samples with &gt, 15% of CD did not differ in motility or viability and acrosome integrity during semen storage. In conclusion, incomplete epididymal sperm maturation impairs the in vitro capacitation ability and promotes sperm destabilization in stored boar semen.

O medicamento Bopriva® tem ação anti-GnRH (GnRH - hormônio liberador das gonadotrofinas) e é utilizado na imunocastração de bovinos. Este estudo teve por objetivo avaliar o efeito do medicamento Bopriva® no perímetro escrotal e na produção e qualidade dos espermatozoides de touros de raça Nelore. Foram utilizados cinco animais da raça Nelore (Bos taurus indicus), com 12 meses de idade, que receberam duas doses de Bopriva® com intervalo de 30 dias, sendo cada dose de 1 mL/animal com 400 μg do produto. Foram realizadas, quinzenalmente, a medida do perímetro escrotal e coleta de sêmen pelo método de eletroejaculação (sete coletas), sendo o sêmen analisado física e morfologicamente. O perímetro escrotal reduziu a partir da 4ª coleta de forma significativa (P=0,0056), a motilidade espermática variou de 20% a 66% (média de 39%), o vigor de 1,0 a 2,8 (média de 1,74), a concentração de 87,8 a 296,2 milhões de espermatozoide por mL (média de 242,9 milhões) e os defeitos totais de 36% a 75,6% (média de 49,51%) com alta incidência de gotas citoplasmáticas, caudas enroladas e cabeças isoladas. O medicamento Bopriva® causa redução do perímetro escrotal e interfere na qualidade do sêmen dos animais da raça Nelore, reduzindo a motilidade do sêmen e elevando a porcentagem de anormalidades espermáticas totais do ejaculado.

Since Kupffer cells/macrophages (KCs/MPs) may be involved in the pathogenesis of autoimmune hepatitis (AIH), this pioneer study was undertaken to evaluate KCs/MPs in pediatric AIH in transmission-electron microscope. Methods Ultrastructural analyses were performed using liver biopsies from 14 children with clinicopathologically diagnosed AIH. Results In all AIH children, ultrastructural findings revealed changes in the cells lining sinusoidal vessels, especially KCs/MPs and endothelial cells. KCs/MPs showed increased phagocytic activity and damaged mitochondria, frequently with accompanying intense fibrosis. In 10/14 AIH patients, the cytoplasm of sinusoidal KCs/MPs contained characteristic glassy droplet inclusions. They were round, sharply circumscribed, and contained homogeneous material and distinct translucent fields. Their ultrastructure was identical with the Russel bodies of plasma cells, which were also found in liver biopsies in the same patients. Conclusion Ultrastructural identification of characteristic cytoplasmic droplets with glassy appearance in KCs/MPs, never before described in AIH, provides a useful novel morphological feature in the diagnosis of this disease.

A reproductive analysis of a captive group of jaguars (Panthera onca; n = 6) at the Santacruz Zoological Foundation in Cundinamarca, Colombia, was conducted by performing a longitudinal, noninvasive, hormonal analysis of estradiol and progestogens in females and of androgens in males. During four seasons, female jaguars confined in solitary were evaluated for ovarian activity and spontaneous ovulation, male jaguars for testicular activity. A second hormonal follow-up was conducted in the females after administration of gonadotropins. Hormones were extracted from fecal samples of three females (n = 3) and two males (n = 2). Estradiol measurements were obtained by RIA and progestogens by enzyme immunoassay. The linear mixed-effect regression showed that there was a significant effect of seasons in the concentrations of estradiol (chi square = 15.97, degrees of freedom = 3, P

P-bodies (PBs) are cytosolic RNP granules that are conserved among eukaryotic organisms. In the past few years, major progress has been made in understanding the biochemical and biophysical mechanisms that lead to their formation. However, whether they play a role in mRNA storage or decay remains actively debated. P-bodies were recently isolated from human cells by a novel fluorescence-activated particle sorting (FAPS) approach that enabled the characterization of their protein and RNA content, providing new insights into their function. Together with recent innovative imaging studies, these new data show that mammalian PBs are primarily involved not in RNA decay but rather in the coordinated storage of mRNAs encoding regulatory functions. These small cytoplasmic droplets could thus be important for cell adaptation to the environment.

We present live births resulting from two separate IVF cycles in a couple in which ICSI was performed with sperm specifically selected for presence of small cytoplasmic droplets. These cycles followed previous cycles using standard sperm selection methods in which very poor embryo development and no pregnancies ensued. The male partner was diagnosed with severe male factor infertility including elevated DNA fragmentation.

Commercial ostrich farming is constrained by the absence of a formal animal recording and evaluation scheme as well as by current farming practices. Artificial insemination may have an important role in overcoming these limitations, but requires a thorough knowledge of sperm morphology. Although the morphological characteristics of normal ostrich sperm have been documented, little information is available on the incidence and structural peculiarities of defective sperm in this species. Semen smears were prepared from the ejaculates of five ostriches (Struthio camelus), stained and evaluated. Defects were observed in 17% of sperm studied. Tail defects constituted the most common anomaly. Various forms of bending were the main tail defect, ranging from gentle to acute bends of the principal piece, Dag-like coiling at the head base, sharp reflexes of the midpiece as well as coiling of the endpiece. In contrast, head defects were comparatively low in frequency, with macrocephalic sperm being the defect most frequently observed in this region. Bent, microcephalic, acephalic and round sperm heads were also noted but were few in number. Cytoplasmic droplets occurred frequently in the fixed smears, either associated with the sperm or as free-lying droplets. A small percentage of sperm with multiple defects was recorded. The incidence of morphologically normal sperm in ostrich semen compared favorably with that reported in emu semen, another commercially farmed ratite. However, the range of defects differed appreciably between the two species. Sperm tail anomalies were the most frequent category in the ostrich, whereas head defects comprised the main grouping in the emu.

Sperm morphometry, in combination with other morphological traits and sperm parameters, can be useful for developing a fertility index. In present study various morphological and morphometric characters of cauda epididymal spermatozoa from 30 house rats were measured. About 100 spermatozoa of each rat with normal morphology and abnormalities like abnormal head, midpiece swelling, coiled tail, bent tail, detached heads and cytoplasmic droplets were counted in different fields and percentage of each was calculated. About 50 spermatozoa of each rat were measured for head length (HL), mid piece length (MPL), tail length (TL), head area (HA) and head perimeter (HP) using Magnus pro analytic software. In cauda epididymal spermatozoa of rat primary abnormalities included abnormal heads and mid piece swelling and secondary abnormalities included cytoplasmic droplet, coiled tails, bent tails, free heads. Variation among the sperm abnormalities was considerably high (>50 %). There was little variation in HL, MPL, TL, HA and HP of cauda epididymal spermatozoa of 30 rats (

The aim of the present study was to compare the influence of cultured epididymal epithelial cells (EEC) from corpus, caput or cauda, oviductal epithelial cells (OEC) and non-reproductive epithelial cells (LLC-PK1) on function and survival of epididymal and ejaculated spermatozoa, in the latter case to determine whether such influence differed between morphologically normal and abnormal spermatozoa. For this purpose, either spermatozoa were directly co-cultured with EEC from caput, corpus, or cauda, OEC and LLC-PK1 cells (experiment 1) or a membrane-diffusible insert was included in these co-cultures (experiment 2). EEC cultured from the three epididymal regions did not differently affect the sperm parameters. Morphologically normal spermatozoa presented a higher ability to bind EEC, OEC, and LLC-PK1 than abnormal spermatozoa with cytoplasmic droplets or with tail/head malformations. Epididymal spermatozoa were more able to bind EEC during the first 24 h of co-culture, while ejaculated spermatozoa presented a higher capacity to bind OEC between 30 min and 3 h of co-incubation. In all cases, the ability to bind to epithelial cells was higher when they were co-cultured with EEC and OEC than with LLC-PK1. After 2 h of co-culture, the viability of epididymal spermatozoa was better maintained when they bound EEC than when they bound OEC. Conversely, the viability of ejaculated spermatozoa was better maintained when bound OEC than when bound EEC after 24 and 48 h of co-culture. Our work, apart from corroborating the involvement of morphologically normal spermatozoa in the formation of sperm reservoir, highlights the importance of direct contact spermatozoa-EEC in maintaining the sperm survival in in vitro co-culture, and also suggests that a specific binding between EEC and epididymal spermatozoa exists.

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa.

The Cantabrian brown bear (Ursus arctos) constitutes an endangered subpopulation of the European brown bear in the north of Spain. We have carried out a post-mortem recovery of epididymal spermatozoa from a Cantabrian brown bear (7 years old, 170 kg; 30 min post-mortem), cryopreserving those recovered from the cauda epididymis (929 × 10(6) spermatozoa, 54% motile, 82% cytoplasmic droplets). For freezing, three extenders based on Test-Tris-Fructose + 4% glycerol were used: (1) 325 mOsm/kg and 10% egg yolk; (2) 430 mOsm/kg and 15% egg yolk; (3) 300 mOsm/kg, Equex-EDTA and 20% egg yolk. After thawing, we obtained higher motility for extender 3 (31%), but extender 2 yielded the highest viability (66.9%) and mitochondrial activity (67.1%). Caffeine stimulation showed that extender two rendered the highest recovery values of post-thawing motility with respect to the fresh sample. In conclusion, epididymal spermatozoa of brown bear can be frozen applying an extender with osmolality similar to epididymal environment.

morphologic comparisons of the live and dead sperm population gives information concerning non-lethal and lethal abnormalities. Many of the abnormalities of the sperm heads and midpieces and in particular enlarged heads belong to the latter group. On the other hand, tapering of the head, cytoplasmic droplets and abnormal tails have littel lethal significance. Depletion of the extra-gonadal sperm reserve does not affect the morphology of neither the living nor the dead spermatozoa. This indicates that no significant abnormalities develop after sperm maturation within the epididymis. Cyproterone acetate was shown to affect midpieces and fails of the living spermatozoa within two weeks of treatment indicating an effect on the epididymal level, whereas abnormal heads increase after four weeks indicating an effect on the germinative epithelium, not before the spermatid stage.

To clarify the physiological changes of sperm morphology in active Thoroughbred stallions during the breeding season, we examined the dismount semen collected from the penile urethra immediately after service. The spermatozoa were analyzed for relationships between the morphology and the stallion's age or the number of services. Seasonal variation was apparent in the rate of the sperm tail abnormalities, spermatozoa with cytoplasmic droplets, appearance of medusa cells, and sperm head length. Area and width of the sperm head correlated negatively with age (P

In this study, feed intake and sperm morphology were evaluated in Ogaden bucks supplemented with either agro-industrial by-products or khat leftover. Thirty-five bucks at about 1 year of age, and 15 +/- 1.5 kg initial body weight, were involved in a 12-week feeding programme that had four diet groups. The control (C) diet comprised grass hay alone, fed ad libitum; treatment 1 (T1) comprised grass hay ad libitum supplemented with a mix of agro-industrial by-products at 1% of body weight (BW); treatment 2 (T2) comprised grass hay ad libitum supplemented with khat leftovers at 1% of BW; and treatment 3 (T3) comprised khat leftover alone, fed ad libitum. Bucks in T1-T3 consumed the highest (p less than 0.001) dry matter (DM), crude protein (CP), organic matter (OM) and gross energy (GE) compared with control. Among the treatment groups, DM, OM and GE intakes were higher (p less than 0.05) in T3 and T2, while CP intake was highest (p less than 0.05) in T1. T1 and T3 improved (p less than 0.001) the percentage of morphologically-normal spermatozoa in comparison with C, with bucks in T3 being best. The proportion of total abnormal sperm head shapes and proximal cytoplasmic droplets was lowest (p less than 0.01) in T1 and T3 and highest in C. Although feeding with T1 improved feed intake and sperm morphology, feeding with T3 showed the highest response. Thus, we conclude that T3 and T1 could be utilized as feedstuff to improve sperm morphology in goats under the smallholder farming system in Ethiopia.

The appearance and incidence of sperm abnormalities was studied in 115 ejaculates, collected periodically over I year covering all seasons from five mature, healthy swamp buffalo (Bubalus bubalis) bulls reared under tropical conditions and serving as the current source of semen for artificial insemination (Al) in Thailand. Light microscopy of stained smears was used to investigate sperm head shape morphology, while unstained wet smears were used to examine other sperm abnormalities. The most commonly found morphological aberrations were pear-shaped spermatozoa, knobbed acrosomes, proximal cytoplasmic droplets, simple bent tails and coiled tails under the head, whose ultrastructure (scanning electron microscopy) corresponded to what has been found in other species of bovidae, including varieties of buffalo. The mean prevalence (as least squares mean SEM) of sperm abnormalities was low (below 15%), corresponding to healthy spermiograms. The younger bulls (less than 10 years old, n = 3) had less abnormalities than the older ones (10.1 +/- 0.6% versus 14.1 +/- 0.8%, P less than 0.001, n = 2), including abnormalities of sperm head shape (1.1 +/- 0.3% versus 3.6 +/- 0.3, P less than 0.001), acrosome defects with knobbed acrosomes (1.1 +/- 0.2% versus 1.2 +/- 0.3%, P less than 0.001), spermatozoa with proximal cytoplasmic droplets (2.7 +/- 0.1 % versus 1.4 +/- 0.2%, P less than 0.00 1), defective mid-pieces (0.2 +/- 0.1% versus 0.3 +/- 0.1%) and abnormal sperm tails (3.1 +/- 0.3% versus 5.7 +/- 0.4%, P less than 0.001). The within-bull effect of the year solely affected the incidence of pear-shaped spermatozoa while the incidences of abnormal contour, variable size of sperm head shapes, abnormal mid-piece and simple bent tail among bulls were affected by ejaculate (week of collection). Interaction between age and ejaculate affected only the prevalence of spermatozoa with proximal cytoplasmic droplets. In conclusion, the types of defects encountered were similar to those found in other bovidae, with a very low prevalence over the year the Al sires were followed through.

In this study an attempt was made to analyse morphological changes in sperm with particular attention to sperm with a cytoplasmic droplet, taking into account the age and breed of the boar. The material for the study consisted of ejaculates of insemination boars of five breeds. Morphological examination of sperm was carried out in 30 randomly selected boars--6 individuals from each breed. The morphology of 500 spermatozoa was evaluated in each slide. The percentage of sperm with normal morphology in the semen of the boars varied between breeds and was dependent in varying degrees on the age of the boar. The primary defects in sperm occurred more frequently in Duroc and Pietrain boars than in other breeds and were more dependent on the age of the boar. The high percentage of primary defects in the sperm of young Duroc boars was determined by the frequency of sperm cells with a proximal cytoplasmic droplet on the midpiece. A particularly high percentage of sperm cells with secondary defects was noted in the initial stage of the reproductive life of the boars. The high number of sperm cells with secondary defects noted in the semen of the Duroc and Pietrain boars and the changes occurring with age in the frequency of secondary morphological defects were mainly determined by the frequency of sperm cells with a distal protoplasmic droplet in the middle piece and to a lesser degree by that of sperm cells with a pseudodroplet.

Semen samples collected postmortem from 142 yearling beef bulls (11-13 months old) of three different breeds (Charolais, Hereford and Simmental) were examined to evaluate the proportion of bulls with mature spermiograms. Before slaughter, testes and epididymides were clinically examined and scrotal circumferences were measured. Aliquots of the cauda epididymal contents taken postmortem were used for sperm morphology examination. Sperm head morphology was studied in dry smears stained with carbol-fuchsine. For each preparation, 500 spermatozoa were counted in each smear under light microscope (x 1000). The presence of proximal cytoplasmic droplets, abnormal acrosomes, detached heads and abnormalities of the midpiece and tail were recorded in wet preparations of formol-saline-fixed spermatozoa. For each preparation, 200 spermatozoa were counted in each preparation under a phase-contrast microscope (x 1000). The abnormalities were classified according to a classification system developed by Bane (1961). Morphological abnormalities were recorded as a percentage of the total number of counted spermatozoa. Criteria for a spermiogram to be considered mature included

Titanium dioxide (TiO 2 ) nanoparticles (TNPs) are widely used commercially and exist in a variety of products. To determine if anatase TNPs (ATNPs) in doses smaller than previously used reach the scrotum after entry in the body at a distant location and induce sperm defects, 100% ATNP (2.5 or 5 mg kg−1 body weight) was administered intraperitoneally to adult males for three consecutive days, followed by sacrifice 1, 2, 3, or 5 weeks later (long-) or 24, 48 or 120 h (short-term exposure). Transmission electron microscopy revealed the presence of ANTP in scrotal adipose tissues collected 120 h postinjection when cytokine evaluation showed an inflammatory response in epididymal tissues and fluid. At 120 h and up to 3 weeks postinjection, testicular histology revealed enlarged interstitial spaces. Significantly increased numbers of terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling-positive (apoptotic) germ (P = 0.002) and interstitial space cells (P = 0.04) were detected in treated males. Caudal epididymal sperm from the short-term, but not a long-term, arm showed significantly (P < 0.001) increased frequencies of flagellar abnormalities, excess residual cytoplasm (ERC), and unreacted acrosomes in treated versus controls (dose-response relationship). A novel correlation between ERC and unreacted acrosomes was uncovered. At 120 h, there were significant decreases in hyperactivated motility (P < 0.001) and mitochondrial membrane potential (P < 0.05), and increased reactive oxygen species levels (P < 0.00001) in treated versus control sperm. These results indicate that at 4-8 days postinjection, ANTP induce structural and functional sperm defects associated with infertility, and DNA damage via oxidative stress. Sperm defects were transient as they were not detected 10 days to 5 weeks postinjection.

Sperm morphology was studied in 302 extensively managed Zebu bulls (aged 1.5-9 years), classified as sound (n = 166) or unsound (n = 136) for breeding, under field conditions in the dry tropics of Costa Rica. Single semen samples were collected by electro-ejaculation and fixed in formol-saline solution immediately after collection. Sperm morphology was determined in the field on wet smears using a microscope equipped with phase-contrast optics, and further determined in the laboratory on airdried smears stained with carbol-fuchsin. The frequencies of sperm abnormalities (such as abnormal acrosome, head, neck, mid-piece, tail, and presence of cytoplasmic droplets) were recorded as a percentage of the total number of counted spermatozoa (400 cells). Zebu bulls considered unsound for breeding showed a higher mean prevalence (p < 0.05) of knobbed acrosomes (4.0 versus 0.9%), head defects [specifically, nuclear invaginations and heads with abnormal shapes and sizes (27.6 versus 4.0%)], abnormal tails (11.2 versus 4.7%), and proximal droplets (8.4 versus 1.6%), compared with bulls considered sound for breeding. In these latter bulls, the abnormality most commonly seen was the presence of single bent tails with an entrapped cytoplasmic droplet (3.0 ± 3.7%). Young Zebu bulls (i.e. bulls under 2 years of age) showed a higher percentage of missing acrosomes, and proximal cytoplasmic droplets, than older sires (12.1 versus 2.4%, and 23.9 versus 3.6%, respectively; p < 0.05), interpreted as an indication of low ejaculation frequency and sexual immaturity, respectively. Bulls with a long scrotum and soft testicular consistency (TC) at palpation showed higher percentages of abnormal sperm heads in the ejaculate than bulls with a normal scrotal length (SL) and a normal TC (32.7 versus 12.8% and 30.7 versus 10.3%, respectively; p < 0.05). In addition, Zebu bulls with a scrotal circumference (SC) ≤ 30 cm showed a higher prevalence of proximal cytoplasmic droplets than bulls whose SC was > 30 cm (9.8 versus 2.6%, p < 0.05). A higher mean percentage of abnormally sized and shaped heads, especially undeveloped and narrow at the base, was more frequently found in stained smears than in unstained samples (26.0 versus 9.9%, p < 0.05), which clearly underlines the importance of using both stained and wet smears when assessing sperm head morphology. However, for a quick assessment of sperm morphology under field, tropical conditions, phase-contrast microscopy provides useful information for the spermiogramme evaluation. Se estudió la morfología de los espermatozoides de 302 toros cebú manejados de forma extensiva (de 1,5 a 9 años de edad), clasificados como sanos (n=166) o no sanos (n=136) para la cría, en condiciones de campo en el trópico seco de Costa Rica. Se recogieron muestras de semen individuales mediante electroeyaculación y se fijaron en solución salina de formol inmediatamente después de la recogida. La morfología de los espermatozoides se determinó en el campo en frotis húmedos utilizando un microscopio equipado con óptica de contraste de fase, y se determinó además en el laboratorio en frotis secados al aire teñidos con carbolfucsina. Las frecuencias de las anomalías de los espermatozoides (como acrosoma, cabeza, cuello, pieza media, cola y presencia de gotas citoplasmáticas anormales) se registraron como porcentaje del número total de espermatozoides contados (400 células). Los toros cebú considerados no aptos para la reproducción mostraron una mayor prevalencia media (p < 0,05) de acrosomas nudosos (4,0 frente a 0,9%), defectos en la cabeza [concretamente, invaginaciones nucleares y cabezas con formas y tamaños anormales (27,6 frente a 4,0%)], colas anormales (11,2 frente a 4,7%) y gotitas proximales (8,4 frente a 1,6%), en comparación con los toros considerados aptos para la reproducción. En estos últimos toros, la anomalía más comúnmente observada fue la presencia de colas simples dobladas con una gota citoplasmática atrapada (3,0 +/- 3,7%). Los toros cebú jóvenes (es decir, los menores de 2 años) mostraron un mayor porcentaje de acrosomas ausentes, y de gotas citoplasmáticas proximales, que los toros de mayor edad (12,1 frente a 2,4%, y 23,9 frente a 3,6%, respectivamente; p < 0,05), lo que se interpretó como una indicación de baja frecuencia de eyaculación y de inmadurez sexual, respectivamente. Los toros con un escroto largo y una consistencia testicular (CT) blanda a la palpación mostraron mayores porcentajes de cabezas de esperma anormales en el eyaculado que los toros con una longitud escrotal (LS) normal y una CT normal (32,7 frente a 12,8% y 30,7 frente a 10,3%, respectivamente; p < 0,05). Además, los toros cebú con una circunferencia escrotal (CS) < o = 30 cm mostraron una mayor prevalencia de gotas citoplasmáticas proximales que los toros cuya CS era > 30 cm (9,8 frente a 2,6%, p < 0,05). En los frotis teñidos se encontró con mayor frecuencia un porcentaje medio de cabezas de tamaño y forma anormales, especialmente no desarrolladas y estrechas en la base, que en las muestras sin teñir (26,0 frente a 9,9%, p < 0,05), lo que subraya claramente la importancia de utilizar tanto frotis teñidos como húmedos al evaluar la morfología de las cabezas de los espermatozoides. Sin embargo, para una evaluación rápida de la morfología de los espermatozoides en condiciones tropicales de campo, la microscopía de contraste de fase proporciona información útil para la evaluación del espermiograma. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria

A simultaneous live/dead and acrosome staining, originally described for domestic mammals, was successfully applied on red deer (Cervus elaphus) and fallow deer (Dama dama) spermatozoa collected from the cauda epididymidis and vas deferens of shot stags. The staining is simple enough for routine application. Seven classes of spermatozoa were distinguished in the smears of frozen/thawed semen samples. Morphology, including cytoplasmic droplets, was evaluated as well. Percentage of live cells with intact acrosomes and with no other morphological aberrations might be a practical index of semen quality.

The objective of this experiment was to determine the effect of proximal droplets on sperm-oocyte binding, zona penetration, fertilization, and the developmental competence of oocytes fertilized by sperm with proximal droplets (PD) in an in vitro fertilization (IVF) and culture system. Frozen semen from three bulls (PD1, PD2 and PD3) with varying proportions of normal appearing sperm with proximal droplets and semen from a normal control bull (C) were used in this experiment. The mean number of sperm bound to the zona pellucida (26.8 +/- 2.0, n=100; 15.2 +/- 1.1, n=100; 16.2 +/- 1.0, n=100) for bulls PD1, PD2, and PD3, respectively, were significantly lower (P0.05) than that of the control bull C (47.4+/- 1.9; n=114). No spermatozoa with PD were found bound to the zona pellucida and this finding was consistent among the three bulls. The percentage penetration of zonae for the bulls PD1, PD2 and PD3 (74%, 74/100; 71%, 71/100 and 69%, 69/100, respectively) were not different than that of bull C (72%, 179/245). Similarly, the mean number of sperm penetrating the zona pellucida (1.43+/- 1.2, 1.24 +/- 1.1 and 1.20 +/- 1.1, for bulls PD1, PD2 and PD3, respectively) were not different than that of bull C (1.45 +/- 1.1). However, fertilization rates (8.8%, 8/90; 16.8%, 16/95; and 10.6%, 11/103, for bulls PD1, PD2 and PD3, respectively) were lower (P0.001) than that of bull C (68.7%; 77/112). Similarly, cleavage rates (5%, 10/200; 8%, 8/100 and 14%, 15/111) for the bulls PD1, PD2 and PD3, respectively, were lower than that of the control bull, C (60.7%; 79/130). Cleaved zygotes resulting from the fertilization of oocytes by apparently normal sperm from bulls with PD did not develop beyond cleavage, whereas, 43.8% (57/130) morulae and 20% (26/130) blastocysts were produced by oocytes fertilized by sperm from bull C. In summary, normal appearing sperm with PD did not bind to the zona pellucida. Apparently normal sperm with out proximal droplets co-existing in the semen along with sperm containing PD were also functionally deficient, resulting in reduced zonae binding and zygotes resulting from insemination with semen with a high percentage of PD did not develop beyond cleavage.

This article describes an undergraduate laboratory that introduces students to ion channels and patch clamp electrophysiology. It is given in conjunction with third year biochemistry lectures on membrane transport proteins and utilises cytoplasmic droplets from Characean algae. These droplets are very easily obtainable (the typical preparation time is around ten minutes), contain abundant channel activity and significantly, readily form the high electrical resistance seals that are required for resolution of single channel events. Most students have been able to observe electrical events that are the result of conformational changes in single protein molecules. When the students are not patch clamping they are set a problem that requires them to devise a model of how several different types of channel might interact to produce a cellular response to osmotic challenge.

Objective: To determine whether varicocele is associated with retention of sperm cytoplasmic droplets in infertile men. Design: Retrospective study. Setting: University infertility clinic. Patient(s): Nonazoospermic men with idiopathic (n = 69) and varicocele-associated infertility (n = 73), and 20 fertile controls presenting for vasectomy. Intervention(s): None. Main Outcome Measures(s): Standard semen parameters and percentage of spermatozoa with cytoplasmic droplets on Papanicolaou smears. Result(s): No statistically significant differences were found between the fertile and infertile groups with respect to semen volume. Fertile controls had significantly greater mean percent sperm motility and normal morphology than infertile men. The mean percentage of sperm with residual cytoplasm was statistically significantly different in all three groups. Infertile men with varicocele had the highest percentage of sperm with cytoplasmic droplets, the next highest level being in men with idiopathic infertility and the lowest level in fertile controls (11.7 ± 1.0, 8.1 ± 0.9 and 3.2 ± 0.4%, respectively, P Conclusion(s): Our data show that idiopathic and even moreso, varicocele-related male infertility are conditions associated with impaired disposal of residual sperm cytoplasm by the testis and/or epididymis. These data provide a possible mechanism for the observed semen abnormalities and reduced fertility potential associated with varicocele and idiopathic male infertility.

Semen characteristics of alpacas were studied after repeated collections. Twelve adult males were divided into three groups of four each for semen collection once, twice, or three times every other day. The duration of copulation; volume of ejaculate; pH; motility; sperm concentration (number of sperm/milliliter semen); total number of sperm per ejaculate; and percentages of live, normal, and abnormal spermatozoa were analyzed by regression analysis. Semen color and consistency were analyzed by the chi-square test. Between the first, second, and third ejaculations, there were differences (p0.05) in sperm concentration; percentages of normal spermatozoa and abnormal spermatozoa; sperm with abnormal heads and abnormal tails; and consistency (viscous, viscous, and semi-viscous). There were no differences (p0.05) in ejaculated volume, percentage of live spermatozoa, pH, percentage of cytoplasmic droplets, and duration of copulation. Some males from which semen was collected on the three-mating schedule ejaculated only seminal plasma during the second and third copulation starting on Day 10 of the study. There were differences between males (p0.05) for most of the characteristics studied. In sum, frequency of mating affected some semen characteristics that may be important determinants of the fertility of male alpacas.

The timing of shedding and disintegration of cytoplasmic droplets from boar and goat spermatozoa was determined. More than 90% of spermatozoa from the cauda epididymidis and ductus deferens had a droplet each at the distal region of the middle piece in both species. In boars, the droplets were still observed in 97% of spermatozoa immediately after ejaculation. After 1 min of ejaculation, however, the percentage of spermatozoa without the droplet greatly increased to 65%, with no significant changes thereafter. Moreover, most of the free droplets shed from spermatozoa had already disappeared after 1 min of ejaculation. In goats, most spermatozoa (90%) had no droplet even immediately after ejaculation, and the rate of free droplets remaining in the semen was 63%. However, within 2 min of ejaculation, the rate of free droplets remaining in the semen dropped to 8%. These results indicate that shedding of cytoplasmic droplets from boar spermatozoa and their disintegration occur within 1 min after ejaculation. They also suggest that droplets are shed from most of goat spermatozoa during their transit through the urethra and/or immediately after ejaculation, and that their disintegration occurs within a few minutes after ejaculation.

Recent studies have shown cytoplasmic droplets to be normal morphological occurrences in human male spermatozoa. When the cytoplasm around the sperm midpiece is present in large amounts, however, pathological effects may transpire. The cytoplasmic droplet then becomes known as excess residual cytoplasm, which can impair overall sperm function and produce higher levels of reactive oxygen species, potentially leading to male infertility. Though the distinction between cytoplasmic droplets and excess residual cytoplasm has been made, some studies fail to recognize the difference and incorrectly label the latter as a cytoplasmic droplet. This review attempts to clarify excess residual cytoplasm’s effect on fertility, examine the enzymes responsible, and suggest tests and possible treatment options for those affected by this defect.

Las gotas citoplásmicas (GCs) son remanentes del citoplasma que quedan adheridos al espermatozoide después de la espermatogénesis, constituyen la anormalidad espermática más frecuente en porcinos, y se relacionan claramente con baja fertilidad. Hay serios indicios de que la fructosa y el AMPc del plasma seminal intervienen en la maduración espermática, en el desprendimiento de las GCs, y en la reacción acrosómica. El objetivo del presente estudio fue evaluar el efecto de la adición de plasma seminal, en el desprendimiento de las GCs en machos con diagnóstico de persistencia de las mismas. En el estudio se emplearon tres verracos (dos con persistencia de GCs y uno normal) de tres a cinco años de edad, alojados en la granja Montelindo de la Universidad de Caldas; a los cuales se les realizaron análisis seminales semanales, completos, durante cuatro meses. Se llevó a cabo un arreglo factorial 3x3x2 (adición a la FR de los machos con persistencia de GCs de 0%, 20% de PSMS y 20% de PSMGCs; 0, 60 y 120 minutos de incubación, y 16 y 37ºC de temperaturas de incubación) en un diseño de bloques completos al azar, analizado mediante análisis de varianza y prueba de Tukey. La incubación del semen de machos con persistencia de GCs con PSMGCs redujo más del 4% las GCs respecto a la incubación sin PS y con PSMS; igualmente se registró reducción de aproximadamente el 5% en las GCs, al aumentar el tiempo de incubación de 0 a 120 minutos, y alrededor de 2% al llevar la temperatura de incubación de 16 a 37ºC. The cytoplasmic droplets (CDs) are remnants of the cytoplasm that are attached to the sperm after spermatogenesis. CDs constitute the most frequent sperm abnormality in pigs and are clearly related to low fertility. There are serious indications that fructose and the seminal plasma AMPc are involved in sperm maturation in the CDs detachment and in the acrosome reaction. The objective of this study was to evaluate the effect of the addition of seminal plasma in the CDs detachment in males with diagnosis of persistence of such detachment. Three boars (two with persistence of CDs and one normal) from three to five years of age, housed in the Montelindo farm at Universidad de Caldas were used in the study. These boars were performed complete seminal analysis weekly during four months. A factorial arrangement 3x3x2 (addition to the males FR with CDs persistence of 0%, 20% of SPHM and 20% of SPMCDs; 0, 60 and 120 minutes incubation, and 16, and 37ºC incubation temperature) was carried out in a randomized complete blocks design, analyzed through variance analysis and Tukey's test. Incubation of males semen with persistence of CDs with SPMCDs decreased more than 4% the CDS with regards to incubation without SP and SPHM; similarly, there was reduction of approximately 5% in CDs when increasing incubation time from 0 to 120 minutes, and about 2% when increasing incubation temperature from 16 to 37ºC.

The objective of this study was to determine the effects of method and clinician on stallion sperm morphology evaluation. Five clinicians evaluated 60 semen samples using wet-mount preparations with phase-contrast, eosin/nigrosin-stained semen smears, and Papanicolaou-stained semen smears. There were significant differences among methods for all sperm morphology categories and most intra-class correlation coefficients were only fair to moderate. The use of wet-mount preparations facilitated detection of acrosome defects, nuclear vacuoles, and cytoplasmic droplets when compared to stained smears. Smearing stallion semen samples onto slides increased the proportion of detached sperm heads. In addition, acrosome defects, nuclear vacuoles, rough/swollen midpieces, and cytoplasmic droplets were difficult to observe with Papanicolaou stain; this method resulted in overestimation of normal sperm when compared to other methods. There were significant differences among clinicians for all sperm morphology classification categories. In conclusion, this study demonstrated that sperm morphology evaluation results varied, depending on the evaluation method and clinician. Wet-mount preparation with phase-contrast microscopy appeared to be more sensitive for identification of abnormal stallion sperm when compared to stained smears. Veterinary andrology laboratories should invest in training, continuing education, proficiency testing, and other quality control measures to minimize the variation of sperm morphology evaluation results among clinicians.

Genital organs of 10 healthy, adult Mithun bulls (6-8 years old) that were slaughtered at the dwellings of tribal people for meat were collected. Immediately after collection, spermatozoa from 3 different regions of the epididymis, i.e. the head, body and tail, were obtained to study morphological changes of the spermatozoa during passage through these regions. The prevalence of proximal cytoplasmic droplets significantly decreased from the head to the tail of the epididymis. Conversely, the percentage of distal cytoplasmic droplets increased significantly from the head to the tail region. The incidence of tailless heads rose significantly from head to body and then reduced significantly in the tail region. The percentage of total head abnormalities did, however, not change markedly, but total mid-piece and tail abnormalities differed significantly between the three epididymal regions.