Proximal Cytoplasmic Droplets Research Articles

Impact of environmental variables on some reproductive metrics of white-lipped peccaries (Tayassu pecari) raised in Atlantic Forest of Brazil

Knowledge on male reproductive physiology is essential for the development of effective conservation strategies. This study investigated the influence of environmental variables on certain reproductive metrics in white-lipped peccaries (Tayassu pecari) raised in the Atlantic Forest. After anesthetization, testicular and cauda epididymis biometry were evaluated in nine adult male individuals subjected to electroejaculation. Semen was evaluated for volume, pH, concentration, total number of sperm, sperm morphology, membrane integrity, and kinematic parameters. Concurrently, environmental variables were collected from the day before, for the previous 14 days (estimated for sperm maturation in epididymis), and the period of 51–55 days (corresponding to the spermatogenic cycle) before semen collection. Overall, it was observed that rainfall is the most important environmental variable influencing the reproductive parameters of white-lipped peccaries, being positively correlated with the amplitude of lateral sperm head displacement (ρ = 0.62, P < 0.05) and the appearance of proximal cytoplasmic droplets in sperm (ρ = 0.62, P < 0.05). In addition, the testicular biometry of the species is influenced by the set of environmental variables of air temperature, rainfall, and relative humidity (ρ ≥ 0.60, P < 0.05). On the other hand, epididymal biometric data showed numerous correlations between cauda epididymis metrics and sperm parameters (ρ = 0.68, P < 0.05). This information will be useful to improving conservation strategies for these animals, contributing to their management in captivity and to reintroduction programs, especially in the Atlantic Forest where the species is declining.

Heat Shock Proteins Are Differentially Expressed in the Domestic Cat (Felis catus) Testis, Epididymis, and Vas Deferens.

Heat shock proteins (HSPs) play key roles in controlling the morphological transformation of germ cells during spermatogenesis and posttesticular maturation of sperm. This study aims to reveal the presence and localization patterns of large adenosine triphosphate (ATP)-dependent HSPs family members in adult domestic cat testis and excurrent ducts utilizing Western blot, immunohistochemistry, and immunofluorescence techniques. The results indicated that the relative amounts of heat shock protein D1 (HSPD1)/HSP60, heat shock protein C (HSPC)/HSP90, and heat shock protein H (HSPH)/HSP105/110 were highest in the testis, while heat shock protein A (HSPA)/HSP70 was highest in the corpus epididymis. HSPs exhibited spermatogenic stage-dependent localization patterns in germ cells. Sertoli and Leydig cells were positive for other HSPs except for HSPC/HSP90. The tubules rectus and rete testis epithelia showed only HSPD1/HSP60 and HSPA/HSP70 immunoreactivity, while the ciliated cells of efferent ductules were positive for all HSPs. In the epididymis and vas deferens, HSPs localizations were cell and region specific. HSPD1/HSP60 was localized in the midpiece of the immature spermatozoa tail, while HSPA/HSP70 and HSP90 were found only in the proximal cytoplasmic droplet (CD). HSPH/HSP105 was observed in CD and the principal piece but not the midpiece. Overall, the different expression of HSPs throughout the domestic cat testis and excurrent ducts indicates their critical roles in maintaining reproductive functions under physiological conditions.

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

Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation-produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been debated by researchers over the last decade. The objective of this study was to evaluate the relationship between various sperm morphologies and ROS production in fresh and cooled stallion semen by employing the novel method of imaging flow cytometry for stallion semen assessment. For evaluation of fresh semen, single ejaculates (n=5) were collected from four resident stallions at the University of California, Davis. For the evaluation of 24-h cool-stored semen, single ejaculates were collected from stallions at Texas A&M University (n=5) and shipped to the University of California, Davis overnight for evaluation. Ejaculate volume, sperm concentration and motility parameters were recorded. Samples were co-stained for viability and ROS detection with SytoxGreen™ and dihydroethidium (DHE), respectively, and evaluated with the Amnis® ImageStream® system (Luminex Corporation). Antimycin, an electron transport chain inhibitor that triggers ROS production (1μM), was used as a positive control for DHE, while dead cells (2× snap frozen in liquid nitrogen) served as a positive control for SytoxGreen™. Unstained samples were also evaluated as controls. Imaging flow cytometric analysis was performed with the ideas® software (Luminex Corporation). Evaluated morphologies included abnormal head (AH), abnormal midpiece (AM), abnormal tail (AT), proximal cytoplasmic droplet (PD), or distal cytoplasmic droplet (DD), and morphologically normal (MN) cells. For fresh semen, an additional abnormality, coiled tail and midpiece (CTM) was assessed; 24-h cool-stored semen did not contain enough viable CTM cells for analysis. Only cells with obvious, single abnormalities were selected for the first portion of analysis to minimize subjectivity. Mixed effects modelling was used to evaluate the relationship between each morphologic classification and the corresponding DHE fluorescence intensity. Compared to the MN population, ROS production was significantly higher in viable cells with AH, PD and AM (p < .0001) in both fresh and cooled semen. CTM cells had significantly higher levels of ROS production compared to MN cells in fresh semen (p < .0001). There was no significant difference in ROS levels between MN cells and AT and DD cells in either fresh or cooled semen (p > .05). These results suggest that ROS generation is indicative of abnormal cell morphology and function and confirm that imaging flow cytometry is a valuable tool for the assessment of stallion semen.

Testicular Ultrasound Analysis as a Predictive Tool of Ram Sperm Quality

Simple SummaryIn animal production, the prediction of male fertility is vital for the success of specific techniques such as artificial insemination. Thus, testicular ultrasound, a non-invasive diagnostic procedure, could be a useful tool. Moreover, recent ultrasound-video analysis and software developments allow the visualization of tissue at the microscopic level. The objective of this work was to establish a possible correlation between testicular ultrasonography and semen quality in rams. For this purpose, the testicles of nine rams were evaluated and the semen was analyzed for one year. The results revealed that the number of white and grey pixels correlated with sperm parameters indicating poor seminal quality. On the other hand, the increase in the seminiferous-tubule density or the lumen area of these tubules was related to a rise in seminal quality. Therefore, ultrasound-video analysis could be a good tool for evaluating the fertility of rams, either for artificial insemination or on the farm.Testicular ultrasound is a non-invasive technique that could be very useful for predicting ram seminal quality. Recent software developments allow macroscopic and microscopic evaluation of testicular parenchyma. Thus, the aim of this study was to evaluate the testicular echotexture using ultrasound-video analysis and investigate its possible correlation with semen quality. Nine rams were evaluated for one year using a portable ultrasound scanner and the echotexture was analyzed with ECOTEXT® software. The number of black (Ec1), white (Ec2), and grey pixels (Ec3), tubular density (TD), lumen area (LA), and lumen diameter (LD) were analyzed. Semen was collected by an artificial vagina the same day and the sperm concentration, morphology, motility, viability, phosphatidylserine (PS) translocation, reactive-oxygen-species (ROS) levels, DNA damage and capacitation state were evaluated. Ec2 and Ec3 correlated positively with “bad quality” sperm parameters (the percentage of spermatozoa with high ROS levels, with PS translocation and proximal cytoplasmic droplets), and negatively with motility. In contrast, TD and LA showed a positive correlation with “good quality” parameters (motility or normal morphology) and a negative correlation with spermatozoa with high ROS levels, with DNA fragmentation, and proximal or distal cytoplasmic droplets. Thus, echotexture analysis by ultrasound-video analysis could be a valuable tool for assessing ram fertility.

Changes of sperm morphology in Duroc, Landrace and Large White boars depending on the ambient temperature during the year

Utilisation of artificial insemination has led to the increased role of male animals. They significantly contribute to the efficiency of productivity; therefore, sperm quality has been emphasised. The aim of this study was to analyse changes in the boar sperm morphology during the year in forty-two Duroc, Landrace and Large White boars in the Czech Republic. For each boar, a spermiogram was evaluated every month in 2018. The number of morphologically abnormal spermatozoa was recorded every month to evaluate the morphology. The abnormalities were categorised as abnormalities of the head, abnormalities of the acrosome, proximal cytoplasmic droplets and abnormalities of the tail. Throughout the study, the temperature in the stable was monitored. Changes in the sperm morphology were recorded in all breeds during the year, the changes were different for each breed. In the Landrace boars, the highest concentrations of morphologically abnormal spermatozoa were observed in July and August (P &amp;lt; 0.05). In this period, the concentration of the tail defects increased (P &amp;lt; 0.05), while, in October, a higher concentration (P &amp;lt; 0.05) of spermatozoa with defects of the acrosome was recorded in the Landrace boars. In the Duroc and Large White boars, the highest morphologically abnormal spermatozoa values were observed in September and October (P &amp;lt; 0.05). In the Duroc boars, the increase in the morphologically abnormal spermatozoa in this period was mainly caused by an increased occurrence of proximal cytoplasmic droplets (P &amp;lt; 0.05). On the contrary, in the Large White boars, the increase was related to a higher incidence of tail defects (P &amp;lt; 0.05). These results show that changes in the sperm morphology are influenced by the ambient temperature and the breed.

Semen characteristics of the three genetic types of boars reared in Benin

Objective: To characterize the semen of three genetic types of boars (local, improved and Large White) reared in Benin. Methods: Semen of local, improved and Large White boars reared in Benin were collected using the gloved hand method and analyzed to determine volume, pH, concentration, mobility, motility, and morphology. The effect of the genetic type of boar on semen characteristics was aslo studied. Results: Duration of ejaculation and semen volume of Large White boar were significantly higher than those of local and improved boars (P&lt;0.05). The semen of improved boars had a higher motility score than that of Large White and local boars (P&lt;0.001). The semen of local boars was more concentrated in the spermatozoa than that of improved and Large White boars (P&lt;0.05). The proportion of spermatozoa of improved boars with normal morphology (93.6%) was significantly higher than that of local (82.2%) and Large White boars (81.6%) (P&lt;0.001). The proportion of spermatozoa with folded tails in the semen of Large White boars (9.2%) was significantly higher than that observed in improved (1.8%) and local (5.0%) boars (P&lt;0.001). The proportion of spermatozoa with proximal cytoplasmic droplets in semen of improved boars (2.7%) was significantly lower than that in Large White (6.8%) and local (9.7%) boars (P&lt;0.001). The local (1.5%) and Large White boars (1.1%) showed more spermatozoa with distal cytoplasmic droplets in their semen compared to the improved boars (0.4%). Conclusions: The semen characteristics of pigs reared in Benin vary from one genetic type to another. Each genetic type has a strong point. The Large White boar produces larger semen, the local boar produces more concentrated semen and the improved boar produces spermatozoa that are morphologically better. The semen of these three genetic types can be used in artificial insemination but the improved boar's semen is more recommended.

MORPHOLOGICAL CHANGES IN EPIDIDYMAL SPERMATOZOA OF RED SOKOTO (MARADI) BUCKS

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 first 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&lt;0.05) lowest at the cauda epididymis. Other morphological changes did not follow a particular trend.

The frequency of morphological defects of epididymal sperm in exotic animal species of the family Bovidae kept in the Czech Republic

Epididymal sperm morphology was evaluated by strict criteria using the DeSMA software. A total of 134 animals from the Dvůr Králové ZOO were included in the study. The aim of this study was to determine the prevalence of morphological defects of epididymal sperm in animals of the familyBovidaein species that are not native to the Czech Republic and are bred here only in zoos. Our results showed that the frequency of abnormalities was higher in exotic species of theBovidaefamily compared to domestic species. Furthermore, the acquired defects prevailed over developmental abnormalities. Cytoplasmic droplets are a characteristic feature of epididymal sperm. Their average prevalence in the wholeBovidaefamily is almost 30%. However, distal droplets are not considered to be sperm pathology and, therefore, they are not the most frequent defect. The most common abnormalities are acquired head defects, followed by acquired tail defects, and then by acquired neck defects, mainly represented by proximal cytoplasmic droplets. The lowest prevalence was found for developmental abnormalities, with heads being more often affected than tails. The remaining defects were sporadic.

Unilateral testicular degeneration in dogs: Effects on spermatozoal characteristics, testis and cauda epididymis

Testicular degeneration is an important cause of poor fertility in dogs, but there is little knowledge on its effects on spermatozoa in affected dogs. The study investigated the specific effects of unilateral testicular degeneration (UTD) on spermatozoal characteristics in the testis and epididymis. Ten sexually mature Nigerian indigenous breed of dogs, comprising 5 normal dogs and 5 dogs with UTD were used for the study. The testis and epididymis were removed via orchidectomy for morphological and histopathological evaluation. Sperm in the testis and cauda epididymis were analysed. The samples were grouped into four as N1 (normal right testis), N2 (normal left testis), ND (non-degenerated testis in UTD dogs), and D (degenerated testis in UTD dogs). Data were analysed using one-way ANOVA. There were significant decreases (p&lt;0.001) in testes weight, length, width and volume, and the gonado-somatic index in the D testes compared to the contralateral ND and the N groups. The D group also had significantly lowered epididymal sperm total and progressive motility, viability and concentration (p&lt;0.001), and a decreased testicular sperm concentration (p&lt;0.01). Moreover, there was a decreased percentage (p&lt;0.001) of morphologically normal sperm, with increased prevalence of sperm abnormalities in the D group compared to the other groups. In comparison with the N groups, the ND group had a significantly lowered (p&lt;0.05) epididymal sperm progressive motility, with increased percentage (p&lt;0.01) of sperm with proximal cytoplasmic droplets and looped tails. The findings demonstrated that UTD in dogs adversely affected spermatozoa in the testis and cauda epididymis. There was also evidence of compromised spermatozoa in the epididymis contralateral to the degenerated testis.&#x0D; Keywords: Canine; Epididymis; Spermatozoa; Testicular atrophy; Testicular degeneration

Effect of senescence on morphological, functional and oxidative features of fresh and cryopreserved canine sperm

The present research aimed to compare the hormonal profile, sperm quality and freezability of young and senile dogs. Dogs were assigned into Young Group (n = 11) and Senile Group (n = 11), additionally divided into Fresh Semen Group and Cryopreserved Semen Group. Males were evaluated for libido score and blood estrogen and testosterone assay. Sperm morphofunctional evaluations were performed based on Computer Assisted Sperm Analysis, morphology, mitochondrial activity, mitochondrial membrane potential, plasma and acrosomal membrane integrity, and DNA fragmentation. Sperm oxidative features were: protein oxidation, lipid peroxidation and production of advanced glycation end-products. Young dogs had higher libido score, sperm velocity average pathway, linearity of motility and mitochondrial activity index and lower percentage of major defects, total defects and proximal cytoplasmic droplet, despite the lack of difference between hormone profile of aged dogs. Fresh semen of senile dogs had increased percentage of spermatozoa with high mitochondrial membrane potential compared to young dogs and to cryopreserved sperm. Cryopreserved semen of young dogs had higher acrosomal membrane integrity compared to the Senile Group. In conclusion, sperm of aged dogs have reduced quality, signaled by higher morphological defects, ultimately altering sperm mitochondrial function and sperm kinetics. Furthermore, spermatozoa from senile dogs are more sensible to cryoinjury.

Microscopic sperm head damage and abnormalities as heat stress indicators in Australian Merino rams &lt;i&gt;(Ovis aries)&lt;/i&gt; in Northern Patagonia, Argentina

In Northern Patagonia, the mating season starts on March 15th, when rams are submitted to summer temperatures. Exposure of rams to heat stress increases the prevalence of microscopic damage to spermatozoa, morphological abnormalities, and reductions in fertility. This study assesses the adaptive capabilities of six unshorn and six shorn Australian Merino rams, half of which were treated in a heat chamber for eight hours for five days, gradually reaching a temperature of up to 40 °C. Microscopic damage, abnormalities and ultramicroscopic alterations of the plasma membrane and the acrosome of sperm head were analysed. There were significant differences in the percentage of tailless spermatozoa and proximal cytoplasmic droplets between post-treatment periods. Temperature primarily affected the shorn rams and the sperm heads during spermiogenesis. Submicroscopic alterations were observed when the plasma membrane was present in the anterior segment. These alterations can be intact, waved, or dilated. When the plasma membrane was absent, the acrosome might be intact, dilated, and waved. In addition, the outer acrosomal membrane may completely lose its contents or have a nude nucleus. The plasma membrane assumes a waved shape as a result of the effect of temperature on the epididymis. According to this study, the tailless head, proximal cytoplasmic droplets, and the ultramicroscopic categories studied were robust indicators of semen heat stress. After ten weeks, the sperm head recovered its normal shape. Unshorn rams are better adapted to summer heat stress than shorn ones. Microscopy and transmission electron microscopy alterations have been shown to be excellent indicators of thermal stress in Australian Merino rams and may be useful tools to help sheep farmers choose when to begin the mating season, which will vary depending on the environmental conditions of the summer.

Relação entre o perímetro escrotal e parâmetros da qualidade do sêmen em machos da raça Guzerá, da puberdade até os 36 meses de idade

O objetivo deste estudo foi verificar a associação entre o tamanho do perímetro escrotal e as características físicas e morfológicas dos ejaculados em 191 touros da raça guzerá. Os touros foram divididos por faixas etárias dos 12 aos 36 meses de idade. As mensurações de perímetro escrotal e medidas testiculares foram obtidas com ajuda de paquímetro e fita métrica de acordo à metodologia do Colégio Brasileiro de Reprodução Animal. Os animais que apresentaram perímetro escrotal ≥ 19 cm foram submetidos à coleta de sêmen utilizando-se estímulo eletro-ejaculatório. Foram avaliadas as características espermáticas dos ejaculados como motilidade, vigor e também realizadas avaliações de concentração espermática e a presença de células arredondadas. A correlação do perímetro escrotal com a motilidade espermática por faixa etária foi positiva r=0.94; (P&lt;0, 005). Neste estudo foi observado que a presenças de espermatozoides com gota citoplasmática proximal, células arredondadas no ejaculado e defeitos de cabeça dos espermatozoides são as características de avaliação objetiva que mais poderiam ser utilizados para seleção de precocidade da produção espermática em machos bovinos. Pode ser concluído que a normalidade da produção espermática na raça Guzerá considerando conjuntamente todos os parâmetros representativos de maturidade do processo da produção espermática foi atingida com idade acima de 28 meses e perímetro escrotal acima de 30 cm.

Physical and morphological characteristics of the first three jets of Pêga jackasses sperm-rich fraction

ABSTRACT The first three jets of the sperm-rich fraction of Pêga jackasses were collected and assessed separately. Five fertile Pêga jackasses were used as semen donors and underwent fractionated semen collection, using an open model artificial vagina. The first three jets of the semen were collected separately and assessed for volume, sperm motility, vigor, concentration/mL of semen, and sperm morphology. These characteristics were compared between first, second and third jets and between jackasses. It was observed that the jet volume differed (P&lt;0.05) between jackasses, although it was similar (P&gt;0.05) between first, second and third jets. Sperm motility did not differ (P&gt;0.05) between jets and jackasses. Vigor was similar (P&gt;0.05) between jets of the same jackass, and only the first jet differed (P&lt;0.05) between jackasses. The first, second and third jets of the sperm-rich fraction had decreased sperm concentrations (P&lt;0.05) of 955.56, 725.56 and 280.56x 106 sperm/mL of semen, respectively. Sperm morphology differed between the first three jets only for the incidence of mid-piece defect, higher in the third one (4.26%), compared to the first (3.36%) and second (3.38%) ones. When comparing the morphological characteristics of the sperm-rich fraction between five jackasses, regardless of the jet, there were differences in the percentage of normal sperm, proximal cytoplasmic droplet, mid-piece and head defects.

Subacute ruminal acidosis reduces sperm quality in beef bulls.

Breeding bulls are commonly fed high-energy diets, which may induce subacute ruminal acidosis (SARA). In this experiment, 8 Santa Gertrudis bulls (age 20 ± 6 mo) were used to evaluate the extent and duration of effects of SARA on semen quality and the associated changes in circulating hormones and metabolites. The bulls were relocated and fed in yards with unrestricted access to hay and daily individual concentrate feeding for 125 d before SARA challenge. Semen was collected and assessed at 14-d intervals before the challenge to ensure acclimatization and the attainment of a stable spermiogram. The challenge treatments consisted of either a single oral dose of oligofructose (OFF; 6.5 g/kg BW) or an equivalent sham dose of water (Control). Locomotion, behavior, respiratory rate, and cardiovascular and gastrointestinal function were intensively monitored during the 24-h challenge period. Rumen fluid samples were retained for VFA, ammonia, and lactate analysis. After the challenge, semen was then collected every third day for a period of 7 wk and then once weekly until 12 wk, with associated blood collection for FSH, testosterone, inhibin, and cortisol assay. Percent normal sperm decreased in bulls dosed with OFF after the challenge period ( < 0.05) and continued to remain lower on completion of the study at 88 d after challenge. There was a corresponding increase in sperm defects commencing from 16 d after challenge. These included proximal cytoplasmic droplets ( < 0.001), distal reflex midpieces ( = 0.01), and vacuole and teratoid heads ( < 0.001). Changes in semen quality after challenge were associated with lower serum testosterone ( < 0.001) and FSH ( < 0.05). Serum cortisol in OFF bulls tended to be greater ( = 0.07) at 7 d after challenge. This study shows that SARA challenge causes a reduction in sperm quality sufficient to preclude bulls from sale as single sire breeding animals 3 mo after the event occurred.

Frequency of Cytoplasmic Droplets Depends on the Breed and Age of Insemination Boars.

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.

Role of residual cytoplasm on oxidative status during sperm maturation in dogs

During maturation sperm cells acquire their fertilizing ability; however, this event also produces reactive oxygen species and as such local antioxidant protection is required. Thirteen epididymis from dogs were used, and sperm samples were collected from different segments of the epididymis (i.e. caput, corpus and cauda). The samples were evaluated for motility and vigor, permeability of plasma membrane, presence of cytoplasmic droplet, acrosome integrity and the activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD). Samples collected from the cauda showed higher motility, vigor and lower permeability of plasma and acrosomal membranes in relation to the corpus and caput, indicating different levels of maturity. The enzyme activity remained unchanged in the samples. A significant positive correlation was observed in the epididymal caput, between distal cytoplasmic droplet and SOD, and a negative correlation, in the epididymal cauda, between proximal cytoplasmic droplet and GPx, indicating the physiological need of the specific antioxidants in these segments.

Development of an in vitro index to characterize fertilizing capacity of boar ejaculates

The aim of this research was the selection of spermatozoa parameters related to boar fertility performance and their combination into an in vitro index. A first set (data set 1) of 36 Pietrain boars with 138 ejaculates from two seasons with 5083 single-sire inseminations from 34 farms was used to determine correlations between in vitro sperm quality parameters and fertility performance. 2970 ejaculates representing a second set (data set 2) served calculation of seasonal and age effects on semen quality. Morphological spermatozoa parameters were estimated manually with a phase contrast microscope on the day of semen collection, whereas mitochondrial activity and viability were analyzed by double-staining with rhodamine123/propidium iodide on day 2 of semen storage using flow cytometry. Sperm motility was tested on day 7 by thermoresistance (TRT) after 30min (TRT1) and 300min (TRT2) incubation at 38̊C using computer-assisted semen analysis (CASA).Correlations revealed four independent sperm quality parameters qualifying as relevant predictors of boar fertility: (i) percentage of spermatozoa with proximal cytoplasmic droplets, (ii) percentage of spermatozoa with active mitochondria, (iii) beat cross frequency of progressively motile spermatozoa in TRT1, and (iv) oscillation measure of the actual path of progressively motile spermatozoa in TRT2. There were no significant effects of sperm concentration, ejaculate volume, and total number of sperm cells per ejaculate on litter size (LS) and on pregnancy rate (PR). Our findings suggest the usefulness of sperm quality parameters based on adjusted range of methods and enable the construction of an in vitro index as a means to predicting boar fertility.

Morphological and acrosomal changes of canine spermatozoa during epididymal transit

BackgroundDuring epididymal transit, functional and structural modifications leading to full maturation enable male gametes to reach, recognize and fertilize the oocytes. In dogs, little is known on the modifications of spermatozoa during the passage in the epididymis. The aim of this study was to describe the motility, morphology and acrosomal patterns of canine spermatozoa retrieved from the epididymis caput, corpus and cauda.ResultsAfter the dilution required for the collection of epididymal content, sperm motility was significantly higher (P <0.0001) in the cauda compared to corpus and caput.Proportions of spermatozoa with normal morphology were significantly higher in corpus (P =0.02) and cauda (P <0.0001) compared to caput. Overall morphological abnormalities of the head and neck/midpiece were similar in the three different epididymal regions. A significantly increased prevalence of tail defects, mainly represented by single bent tails, was observed in the corpus compared to caput (P <0.0001) and cauda (P =0.006).Numbers of immature sperm with cytoplasmic droplets decreased from the proximal to the distal region of the epididymis. Particularly, proximal cytoplasmic droplets were more frequently found in spermatozoa collected from the caput epididymis than in the corpus (P <0.0001) and in the cauda (P <0.0001), whereas the occurrence of distal cytoplasmic droplets was higher in the corpus than in the caput (P =0.0003) and in the cauda (P <0.05).Significantly higher proportions of spermatozoa with intact acrosomes were retrieved from the cauda epididymis than from the caput (P =0.03) and the corpus (P =0.008). This difference was mainly due to a lower proportion of spermatozoa with abnormal acrosomes (mainly swollen acrosomes) rather than with absent acrosomes.ConclusionsCanine spermatozoa undergo several modifications in the epididymis. The acquisition of progressive motility, migration of the cytoplasmic droplet and acrosomal reshaping lead to mature spermatozoa which are then stored in the cauda epididymis. From this site, spermatozoa can be retrieved and used in assisted reproductive techniques as a valuable tool for propagating genetic traits of high value individuals that dies accidentally or undergoes orchiectomy for medical purposes. Further investigations should be also focused on the potential use of spermatozoa recovered from other epididymal regions.

Impact of proximal cytoplasmic droplets on quality traits and in-vitro embryo production efficiency of cryopreserved bull spermatozoa

BackgroundProximal cytoplasmic droplets (PCDs), a remnant of germ cell cytoplasm, are common non-specific morphological defects in bovine semen. This study evaluated the effect of higher percentages of PCDs on the quality of frozen-thawed bovine semen, embryo production and early embryo development.MethodsThree ejaculates from each of five (group 1: PCD ≤ 1%, control) and eight adult Bos indicus bulls (group 2: PCD ≥ 24%) were analysed. Semen samples were examined for: post-thaw motility, vigour of movement, concentration, sperm morphology, slow thermoresistance test (STT), membrane integrity, acrosome status, mitochondrial function using fluorescent probes association (FITC-PSA, PI and JC-1) and sperm chromatin integrity using acridine orange assay. Two bulls from group 2, with 28.5% and 48.5% PCD, respectively, and three bulls from the control group, each with 0% PCD, were selected for IVF (in vitro fertilisation).ResultsSemen analyses revealed a significant correlation (P < 0.01) between increased rates of PCD and sperm quality traits. Nevertheless, no differences were observed in sperm motility and vigour either before or after the STT or in the percentage of intact acrosomes (analysed by differential interference contrast microscopy (DIC) after STT), but membrane integrity, acrosome status (evaluated with FITC-PSA staining method after thawing) and mitochondrial function were reduced, when compared with group 1 (P < 0.05). The higher incidence of PCD was positively correlated to chromatin damage, especially after three hours of incubation at 37°C. IVF showed similar results for bull C2 (group 1, control) and bull P2 (group 2, group with higher PCDs).ConclusionHigher PCD levels influenced spermatozoa quality traits. IVF and embryo development data showed that cleavage, blastocyst formation and blastocyst hatching may have been influenced by the interaction of morphology traits and individual bull effects.

Differences between epididymal and ejaculated sperm characteristics in donkey

Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymal canal. In the epididymal caput and corpus spermatozoa undergo several biochemical and metabolic changes while the cauda of the epididymis should be considered as the primarily site for storage of the spermatozoa. In the horse spermatozoa from cauda epididymis were collected and frozen, and the fertility of semen assessed. However, no studies have detailed semen characteristics of spermatozoa collected from the cauda epididymis in the jackass. In this study sperm characteristics of spermatozoa in the cauda epididymis of the donkey was reported and a comparison with ejaculated spermatozoal characteristics was performed. Samples from 10 Martina Franca jackasses were collected and analyzed for viability (Propidium iodide/Sybr-14 ® fluorescent stain), mitochondrial activity (Mitotraker ® fluorescent stain), objective motility characteristics (by Computer Assisted Sperm Analyzer – CASA) and morphology. A higher viability and mitochondrial activity in the cauda epididymis samples were reported in this paper. Samples reported in this paper were identified and the percentage of total and progressive spermatozoa was comparable, but trajectories were more rapid (higher VCL) with less progressiveness (higher ALH and lower STR and LIN) in the cauda epididymis. Sperm morphology showed a pronounced variability between jackasses, with comparable values for all morphological subclasses. In this study the loss of the distal cytoplasmic droplets happen close to or after ejaculation because the percentage fell to nearly 0% after ejaculation. As suggested for bulls, the presence of a similar percentage in sperm with proximal cytoplasmic droplet in epididymal and ejaculated semen is likely to indicate a failure in the maturation process.