Sperm Morphology Characteristics Research Articles

Sperm collection in the domestic cat: A comparison of two techniques

Semen collection in cats in the clinic setting can be difficult. However, semen analysis is vital when evaluating breeding soundness of a male. Electroejaculation (EEJ) is currently the most reliable semen collection method but requires specialized equipment and training of the operator. Chemical ejaculation followed by urethral catheterization (UC) is a technique that allows semen collection without special equipment: a catheter is placed into the urethra of a sedated tom and semen is collected passively into the catheter. Earlier studies used the sedative medetomidine at high doses for this procedure. However, medetomidine has been replaced with dexmedetomidine in some countries. This study sought to compare the results of EEJ and UC for semen collection in the domestic cat using dexmedetomidine, a potent α2-adrenoceptor agonist (α2A), as a substitute for medetomidine at the equivalent dose to that used in earlier studies. Twelve domestic cats were collected thrice at weekly intervals. All cats received intramuscular ketamine (5 mg/kg) and intramuscular dexmedetomidine (30 μg/kg) for initial cleanout via EEJ, then randomly underwent either EEJ or UC one week apart. The EEJ was performed under the same anesthetic protocol as the initial cleanout. The UC was performed using intramuscular dexmedetomidine at a dose of 60 μg/kg. Success of collection, total sperm number, sperm morphology, and motility characteristics were analyzed. Sperm was collected successfully from all 12 cats via EEJ and from 11/12 via UC. There were no significant differences in the percentage of total motile, progressively motile, or morphologically normal sperm between ejaculate types when averaged across all cats or individual cats. Although UC yielded a lower volume and higher concentration ejaculate, it consistently produced a lower total sperm number than ejaculates retrieved via EEJ (17.91 x 106 total sperm for UC versus 46.51 x 106 total sperm for EEJ). These results indicated that dexmedetomidine is a very effective sedative and performed satisfactorily in both procedures at the doses used in this study. It was also safe with no adverse effects on healthy toms. EEJ remained the most reliable in terms of assessing semen quality and retrieving semen with adequate number of sperm for breeding purposes. However, UC with dexmedetomidine at this dose demonstrated a 92 % success rate, presenting itself as a remarkably consistent alternative.

Improving chilled and frozen buck sperm characteristics by adding melatonin and L-carnitine to the preservation medium.

This study evaluated the effects of melatonin (MLT) and L-carnitine supplementation on sperm quality and antioxidant capacity during chilled and cryopreservation. Twenty-four ejaculates were collected from six Damascus bucks, 4 ejaculates each, from mid-September to mid-October 2022. The pooled semen from each collecting session was divided into 5 equal aliquots after being diluted (1:10) with Tris-citric acid egg yolk extender. The first aliquot served as a control (treatment-free). MLT was added to the second and third aliquots at low and high doses (LD: 4 and HD: 8 μL/mL) (v/v), respectively, while L-carnitine (LC) was added to the fourth and fifth aliquots at the same aforementioned doses. The aliquots were stored at 4°C for 48 h to assess sperm physical and morphological characteristics, alongside lipids peroxidase (LP) production and glutathione peroxidase (GPX) activity. The optimum doses of MLT and LC that showed potential for maintaining sperm characteristics throughout the chilled storage period were further investigated for protecting the spermatozoa after exposure to cryopreservation stress compared to the control. The results showed higher sperm motility (%) in the MLT-HD group, higher (p ≤ .05) sperm viability (%) in the MLT-LD, and both aliquots of LC at T24 hours of chilled preservation. Normal sperm (%) was higher (p ≤ .05) in both LC-LD and MLT-LD groups than other groups, while sperm acrosome integrity (%) was higher (p ≤ .05) in the LC-LD group. Morphological abnormalities (%) were improved (p ≤ .05) in all treated aliquots compared with control. The mean value of GPX activity was higher (p ≤ .05) in both MLT groups, while the concentration of LP increased (p ≤ .05) in the LC-HD or control groups. Furthermore, supplementing buck sperm medium with 4 μL/mL of MLT or LC improved (p < .05) the sperm characteristics and decreased (p < .05) DNA fragmentation index after thawing.

The role of sex hormones and ROS values in the control of andrology in male double-spurred Francolin (&lt;i&gt;Francolinus bicalcaratus&lt;/i&gt;)

Background: This study explored the relationship between andrology, seasonal sex hormone fluctuations, and ROS values in adult male Francolinus bicalcaratus while establishing baseline parameters.&#x0D; Methods: Double-spurred Francolins (n=5 per season) were randomly selected in both dry and rainy seasons from their natural habitat. They were carefully stabilized with dewormer, antibiotics, and multivitamins, then acclimatized for two weeks at the Experimental Animal House, University of Ibadan, Nigeria. The birds were weighed, sedated, and 3 to 5 ml of blood was collected from the jugular vein in lithium heparinized bottles for serum sex hormone analysis. Testes were excised, weighed, washed with a 1.15% KCL solution, and processed for complete andrology and oxidative stress assays.&#x0D; Results: Sperm count, activity, and morphological characteristics peaked during late rainy seasons, coinciding with increased mating and hatchability. This correlated with high serum testosterone and low ROS titres in the testes. Conversely, early dry seasons witnessed declines in serum testosterone, sperm parameters, and hatchability due to increased ROS titres from food scarcity. Late dry seasons saw further declines in sex hormone levels and elevated ROS titres, leading to the absence of sperm cells.&#x0D; Conclusion: This study highlights how sex hormone and ROS titres influence sperm cell viability in double-spurred Francolins, with significant seasonal variations. These factors mainly support spermatogenesis and fertility during the breeding season, characterized by food abundance and cover. Dry season sex hormone values serve as baselines, while wet season sperm count and ROS levels represent baseline data for this bird species.

Effects of sperm processing techniques on IVF pregnancy rates: a mini-review.

Many factors associated with assisted reproductive technologies significantly influence the success of pregnancy after in vitro fertilization (IVF) either directly or indirectly. These factors include sperm processing techniques, egg retrieval, intrauterine artificial insemination, intracytoplasmic sperm injection, and embryo transfer. Among these technologies, sperm quality is one of the most critical factors for a successful IVF pregnancy. The method used for sperm processing plays a crucial role in determining the quality of sperm. Several widely used sorting techniques, such as conventional swim-up, density gradient centrifugation, magnetic activated cell sorting, and hyaluronic acid, have been extensively compared in various studies. Previous studies have shown that each sperm processing method causes varying degrees of sperm damage, particularly in sperm motility, concentration, morphological features, viability, and DNA integrity. However, sperm processing techniques have been developed slowly, and the impact of these methods on pregnancy rates is still unclear. Further exploration is needed. In this review, we aim to compare the results of different sperm processing techniques concerning sperm quality and IVF pregnancy rates. We will also discuss possible clinical approaches, such as microfluidics and integrated approaches, for testing and improving sperm quality.

Effects of reactive oxygen species, lipids peroxidation, and total antioxidant capacity on sperm morphological functional characteristics and quality embryological development ICSI outcomes

Effects of reactive oxygen species, lipids peroxidation, and total antioxidant capacity on sperm morphological functional characteristics and quality embryological development ICSI outcomes

LACTATIONAL EXPOSURE OF METHOXYCHLOR IMPAIRS SPERM QUALITY IN BALB/C MICE

Numerous pesticides derived from human activity are now disseminated in the environment that have been identified as an estrogenic activity that mainly affects the male reproductive system. The objective study was to determine the dose of methoxychlor (MXC) pesticides administered to the mother during the lactation period that would affect the sperm quality of the offspring in mice. Twenty-four BALB/c strains were divided into control and treatment groups. MXC was administered once daily by intraperitoneal to female mice for days 1 to 21 of lactation periods, with 0.14 mg/g, 0.28 mg/g, and 0,42 mg/g doses of MXC. Male litters were killed at postnatal day (PND) 60, and the cauda epididymis was used to observe sperm motility and morphology characteristics. Parameters observed were the number of spermatozoa abnormal and the percentage of progressive spermatozoa. ANOVA showed that the effect of MXC was significantly different in the case of the number of abnormal sperm and the rate of progressive sperm parameters. Duncan's Multiple Range Test (DMRT) showed that D3 (0.42 mg/g) gave the highest effect of MXC on increasing abnormal spermatozoa morphology and decreasing the percentage of motile sperm. These results indicate that exposure to MXC, which showed a physiological response in the lactation period, reduces the sperm quality of offspring

EFFECT OF VITAMIN E SUPPLEMENTATION ON SPERM MORPHOLOGY AND SEMEN CHARACTERISTICS OF RABBIT BUCKS

Twenty mixed breed rabbit bucks aged between 6–8 weeks with an average weight of 861g were randomly assigned to four (4) dietary treatments with five (5) bucks per treatment in a Completely Randomized Design to determine the effect of feeding graded levels of Vitamin E on sperm morphology and semen characteristics of rabbit bucks. The rabbits received graded levels of vitamin E/kg feed (0mg/kg, 20 mg/kg, 40 mg/kg and 60 mg/kg for treatments 1, 2, 3 and 4, respectively). Feed and water were given to the rabbit bucks ad libitum during the experiment. The semen collected was taken to laboratory for sperm morphology and semen characteristics and the data collected were analysed using general linear model procedure of statistical analysis system (SAS). The results revealed that the mean number of normal cells, detached head and mid piece defect were not significantly (P&gt;0.05) different across the treatments. However, the percent abnormal defects percent of free tail and coiled tail were significantly (P&lt;0.05) lower for rabbits fed 20 and 60 mg/kg and the least bent tail was obtained in 60 mg/kg diet. The results for semen characteristics had significant (P&lt;0.05) differences in all the parameters except for semen volume and pH. The results showed that those fed 60 mg/kg of vitamin E diet gave significantly (P&lt;0.05) higher sperm motility, sperm concentration, live ratio and low dead ratio (87.50 %, 232x106, 87.50 % and 12.00 %) respectively. Therefore, supplementation of vitamin E at 60mg/kg in the diet of rabbit bucks is recommended

INVESTIGATION OF GLOBOZOOSPERMIA’S MORPHOLOGICAL STRUCTURE AND DNA FRAGMENTATION IN OLIGOZOOSPERMIA CASES IN INFERTILE MALES

Total globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa without acrosomes. It is still unclear whether patients whose ejaculate contains both normal and globozoospermic cells (partial globozoospermia) suffer from a variation of the same syndrome. Affected men may experience decreased fertility and even infertility. In some cases, an increased number of cells with DNA fragmentation has also been observed in patients with globozoospermia..In this study, standard semen analysis methods in accordance with WHO criteria were applied to infertile male patient groups consisting of 20 normozoospermic and 20 oligozoospermic individuals who were admitted to our clinic. Age, sperm parameters (volume, vitality, concentration, total motility and morphology) were determined and statistically analyzed in normozoospermic and oligozoospermic infertile men.Sperms were stained with the Eosin-Nigrosin method and were visualized under an immersion lens light microscope and evaluated for vitality. The slides were stained using sperm staining solutions with the Spermac technique and the sperms were evaluated morphologically. Sperm DNA fragmentation damage was evaluated by acridine orange staining method.Our results revealed that sperm morphological features (Kruger test) and sperm DNA fragmentation, obtained with various staining techniques, are important in the clinical approach to male infertility and ART methods, and should be used together.

Histological investigation of the protective effect of metformin on testis and sperm parameters in obese rats with type 2 diabetes mellitus

Background/Aim: Diabetes mellitus (DM) is thought to have adverse effects on the male reproductive system. Metformin (MTF) is a widely used drug in the treatment of DM. This study hypothesized that MTF can reduce the harmful effects of DM on the male reproductive system in addition to diabetes treatment. Methods: Twenty-one adult (15-18 g, 11-12 months) C57BL6 male mice were randomly assigned to three groups, as follows: The Control group (C), Diabetes group (D), and Diabetes + Metformin (D+MTF) treatment group. Groups D and D+MTF were fed a diet composed of 60% fat for four weeks. DM was created by administering a single dose of 30 mg/kg streptozotocin intraperitoneally (i.p.). MTF was given through gavage at a dose of 300 mg/kg/day. At the end of the experiment, sperm parameters were evaluated in the testicular tissue. Histomorphology features and immune expressions of VEGF, Caspase-3, and Ki-67 were evaluated in testicular tissue sections stained with Hematoxylin and Eosin (HE). Results: Sperm concentration, motility, and morphological characteristics of diabetic mice were significantly reduced compared to control, and MTF-treated mice. Seminiferous tubule diameters and Johnsen scores were significantly higher in the control and D+MTF groups when compared to the diabetic group. VEGF and Ki-67 immune expression were significantly higher in the control and D+MTF groups compared to the D group (P=0.001, and P=0.002, respectively). Because of D, Caspase-3 positive cell density was higher in the D group compared to the control and D+MTF groups (P=0.002). Conclusion: Diabetes mellitus had adverse effects on sperm parameters and the testicular tissue. High glucose levels with decreased VEGF immune expression damaged spermatogenic cells and decreased proliferation and differentiation due to impaired intratesticular vascularity. MTF treatment reduced the damage in spermatogenic cells and contributed to the differentiation and division of cells through the restoration of vascularization.

Ethyl acetate fraction of Spathodea campanulata (Bignoniaceae) attenuates lead acetate induced testicular toxicity in male Wistar rats.

The use of Spathodea campanulata in folklore medicine for the management of reproductive disorders has been poorly reported. We sought to investigate the protective potential of the ethyl acetate fraction of S.campanulata stem bark extract (EFSC) on lead acetate-induced (LA) testicular toxicity in male rats. Animals during a 28days treatment received dimethyl sulfoxide (DMSO, 0.1%), LA (20mg/kg), and EFSC (200mg/kg). Others received EFSC only (100, 200, and 400mg/kg) or vitamin E (100mg/kg) 1h prior to LA (20mg/kg) administration. LA administration decreased sperm counts and motility by 36.39 and 40.69% respectively in rats. Also, LA-untreated rats showed elevated malondialdehyde (MDA) and decreased total proteins in testis (260, 33%) and epididymis (62, 29%) respectively. However, EFSC (100, 200, or 400mg/kg) administrations improved sperm morphological characteristics as well as antioxidant status in LA-treated rats. EFSC (400mg/kg) showed improved testis seminiferous tubules that were almost normal in the LA-treated rats. Further, EFSC contains a high 9-octadecenoic acid methyl ester. Overall, evidence by LA-induced testicular toxicity, EFSC provides chemopreventive roles via antioxidant mechanisms.

Correlation of Prostatic Artery Blood Flow Assessed by Doppler Ultrasonography with Semen Characteristics in Beagle Dogs.

Simple SummaryThis study examined the blood flow parameters of the prostate artery before dog ejaculation assessed by pulsed-wave Doppler ultrasound (PwD) changes. The main objective of this study was to characterize the vascular parameters of the prostatic artery in beagle dogs and to explore the potential use of Doppler ultrasonography as a complementary method in dog stud selection. These findings report differences between blood flow parameters in cranial and caudal locations of the prostatic artery in comparison to subcapsular and parenchymal locations. There was a high positive correlation of peak systolic velocity with total volume and sperm concentration. In conclusion, estimation of the PwD parameters of the cranial prostatic artery may be useful in the breeding evaluation of dogs.Pulsed-wave Doppler ultrasonography (PwD) is a method used to rapidly and noninvasively assess blood flow dynamics of the canine prostate. Modifications in gland vascularization can affect seminal plasma production and consequently sperm quality. The aim of this study was to determine the normal blood flow parameters of the prostate artery in beagle dogs and to analyze the correlations between vascular flow and semen quality characteristics. PwD was performed on five beagle dogs (5–6 years) measuring vascular features in four different locations of the prostatic artery (cranial, subcapsular, parenchymal and caudal); the measured features were peak systolic velocity (PSV), end-diastolic velocity (EDV), resistive index (RI) and pulsatility index (PI). Ejaculates were obtained using digital manipulation and semen quality was evaluated by determining macroscopic (total volume, sperm-rich fraction volume, color and pH) and microscopic (sperm motility, morphology, viability and acrosome integrity) characteristics. The values of PSV, PI and RI in cranial and caudal prostatic arteries were significantly higher than in subcapsular and parenchymal arteries (p < 0.05). Moreover, a positive correlation of PSV value in the cranial region of the prostatic artery with total ejaculate volume (p < 0.01, r = 0.612) and sperm concentration (p < 0.01, r = 0.587) was determined. PI index was negatively correlated with sperm concentration (p < 0.01, r = −0.709). In conclusion, the results suggest that the prostatic artery blood flow parameters can affect macroscopic semen quality characteristics in healthy dogs.

Computer-Assisted Analysis of Human Semen Concentration and Motility

Computer-assisted semen analysis systems insist on evaluating sperm characteristics. These systems afford capacity to study and evaluate sperm statistical and morphological characteristics such as concentration, morphology, and motility, which have an important role in diagnosis and treatment of male infertility. In this paper, the proposed algorithm allows the assessment of concentration and motility rate of sperms in microscopic videos. First, enhancement process is required because of microscopic images limitations such as low contrast and noises. Then, for true sperm recognition among noise and debris, a hybrid approach is proposed using a combination between segmentation techniques. After, the use of geometric features of the bounding ellipse of the sperm head led to define sperm concentration. Finally, inter-frame difference is applied for motile sperm detection. The proposed method was tested on microscopic videos of human semen; the performance of this method is analyzed in terms of speed, accuracy, and complexity. Obtained results during the experiments are very promising compared with those obtained by the traditional assessment, which is the most widely used and approved in the laboratories.

Cryopreservation of human spermatozoa with penetrating and non-penetrating cryoprotectants

Сryopreserved spermatozoa are widely used in infertility treatment by assisted reproductive technologies. However, the spermatozoa survival rate remains low in patients with oligoastenoteratozoospermia. Therefore the development of effective cryopreservation methods for spermatozoa from pathospermia is relevant. The effectiveness of cryopreservation spermatozoa from oligoastenoteratozoospermia man using penetrating and non-penetrating cryoprotectants was compared. Sperm motility, viability and morphological characteristics were evaluated after cryopreservation with glycerol and polyvinylpyrrolidone. The average number of spermatozoa count in fresh ejaculate was (11.0±0.2) mln/ml. After isolation of active motile fraction the number of cells was (3.8±0.3) mln/ml and (84.3±8.4) % from them were motile (group 3). (78.8±6.6) % of spermatozoa cryopreserved with glycerol (group 1) and (41.4±8.1) % cryopreserved with polyvinylpyrrolidone (group 2) remained active motile. The spermatozoa viability after cryopreservation was (82.1±8.6) % and (89.6±8.6) % in group 1 and 2, respectively. Despite the high rate of spermatozoa survival in group 1 the number of motile cells decreased to (27.3±4.8) % after cryoprotectant removing stage. Morphological analysis revealed that the incidence of spermatozoa head abnormalities was (25.97±2.67), (19.21±2.67) and (20.57±1.19) % in group 1–3, respectively. The differences of spermatozoa midpiece and tail abnormalities in the study groups were statistically insignificant. The use of polyvinylpyrrolidone as a cryoprotectant allows preserving 90 % of survived spermatozoa from oligoastenoteratozoospermia men after freeze/thawing. The set of spermatozoa head, neck and midpiece abnormalities is significantly lower after cryopreservation with polyvinylpyrrolidone compared with routine method with glycerol. Two-stage spermatozoa cryopreservation method with polyvinylpyrrolidone is promising for assisted reproductive technologies since spermatozoa can be used immediately after warming for oocyte fertilization without cryoprotectant removing step.

Certain morphological characteristics of the spermatozoon used for intracytoplasmic sperm injection (ICSI) are associated with a higher likelihood of developmental arrest and embryo degeneration

Around 25% of in-vitro cultured human embryos arrest their development and degenerate during the first 5 days. The functional and morphological characteristics of the spermatozoa, selected for the intracytoplasmic sperm injection (ICSI) could account for the subsequent embryo development failure. The objective of the present study was to assess the relationship between the morphological characteristics of the spermatozoon used for ICSI and the increased risk of developmental arrest and embryo degeneration. Prospective observational study. A total of 83 spermatozoa from 68 men used in ICSI procedures were studied. Inclusion criteria were: 1) patient age < 39 years, 2) patient body mass index (BMI) < 25 kg/m2, 3) excellent quality oocytes. To find out which sperm morphological features could be responsible for the embryo degeneration the morphology of each individual spermatozoon used for ICSI was recorded. The morphology of all individual spermatozoa was evaluated using high magnification (×6100) immediately before the injection. Seven sperm morphological parameters and the presence of sixteen head, neck and tail abnormalities were evaluated. All 83 embryos were cultured in-vitro in Global one-step medium. The frequencies of occurrence of sperm morphological defects were compared between the resulted viable and degenerated embryos. Data was analyzed using the IBM SPSS software, v.21.0. Among 83 ICSI performed, 63 embryos (75.9%) had successful development and 20 embryos (24.1%) arrest their development before day 5. The incidence of pyriform head, thick midpiece and cytoplasmic residues among the spermatozoa that resulted in embryos with developmental arrest were significantly higher in comparison to spermatozoa that led to successfully developed embryos (20% vs. 13%, 40% vs. 21% and 20% vs. 6%, respectively, p<0.05, Chi-square test). In addition, the spermatozoa resulted in embryos with successful development had significantly shorter heads and tails in comparison with those that resulted in embryo development arrest (4.46 vs. 4.63 μm and 41.42 vs. 43.65 μm, p<0.05, Student t-test, respectively). When receiver operating curve (ROC) analysis was performed to find the optimum cut-off value to predict embryo development sperm head length had the best AUC (AUC = 0.66, CI 95%: 0.51–0.76). The optimum cut-off value for head length was 4.25 μm (sensitivity 89.5%, specificity 60.3%). The recorded spermatozoa tail length had lower AUC (0.63, CI 95%: 0.49–0.77) and the optimal cut-off value of 42.5 μm (sensitivity 63.2%, specificity 42.5%). Specific sperm morphological characteristics, such as head length and tail length and the presence of certain sperm abnormalities (pyriform head, thick midpiece and residual cytoplasmic droplets) are associated with an increased risk of developmental arrest and embryo degeneration. Selection of human spermatozoa with head length < 4.25 μm, tail length < 42.5 μm and without pyriform head, thick midpiece and cytoplasmic residues significantly increase the chance for successful embryo development.

Sperm morphology in relation to semen cation concentration in Yankasa rams

The morphological appearance of sperm cells is a major criterion in semen evaluation. The optimal production of semen of high biological value is influenced by numerous factors. Therefore, a study was conducted to evaluate the relationship between sperm morphological characteristics and semen cation concentrations in 62 rams of Yankasa breed of sheep. Semen samples were collected from each animal on weekly basis for 52 weeks with electro ejaculator. The sperm morphological characteristics examined were: detached mid-piece and tail (DMT), detached head (DH), mid-piece droplet (MPD, coiled and bent tail (CBT) and acrosomal abnormality (ACR). The semen cation parameters measured were: sodium + + 2+ ion (Na ), potassium ion (K ) and calcium ion (Ca ). Pearson correlation matrix of SAS was used to determine the relationships among and between the measured characteristics. The result of the study showed that correlations among the sperm morphological characteristics were low and not significant except that between DH and DMTwhich was perfect (p&lt;0.01; r = 1.00). On the other hand, semen cation concentrations were positively correlated but not + significant among themselves except the correlation between Na and K (p&lt;0.01; r = 0.37) which was significant. Relating the semen cation concentration with sperm morphological + characteristics, it was discovered that potassium ion (K ) measured was negatively and significantly correlated with all the morphological attributes (DMT &amp; DH, r= -0.36 @ p&lt;0.01; CBT &amp; ACR, r= -0.38 @ p&lt;0.05; MPD, r= -0.32 @ p&lt;0.05). It was concluded that + + DH is highly associated with DMT while Na concentration was an indicator for K level in the semen as shown in the result. Also, the higher the potassium ion in semen, the lower the level of sperm morphological abnormalities and vice versa in Yankasa rams.

Nanotechnology-based approach for safer enrichment of semen with best spermatozoa

BackgroundAdvances in nanotechnology have permitted molecular-based targeting of cells through safe and biocompatible magnetic nanoparticles (MNP). Their use to detect and remove damaged spermatozoa from semen doses could be of great interest. Here, MNP were synthesized and tested for their ability to target apoptotic (annexin V) and acrosome-reacted (lectin) boar spermatozoa, for high-throughout retrieval in a magnetic field (nanoselection). The potential impacts of nanoselection on sperm functions and performance of offspring sired by sperm subjected to nanoselection were determined. Fresh harvested and extended boar semen was mixed with various amounts (0, 87.5, and 175 μg) of MNP-conjugates (Annexin V-MNP or Lectin-MNP) and incubated (10 to 15 min) for 37 °C in Exp. 1. In Exp. 2, extended semen was mixed with optimal concentrations of MNP-conjugates and incubated (0, 30, 90, or 120 min). In Exp. 3, the synergistic effects of both MNP-conjugates (87.5 μg – 30 min) on spermatozoa was evaluated, followed by sperm fertility assessments through pregnancy of inseminated gilts and performance of neonatal offspring. Sperm motion, viability, and morphology characteristics were evaluated in all experiments.ResultsTransmission electron microscopy, atomic force microscopy, and hyperspectral imaging techniques were used to confirm attachment of MNP-conjugates to damaged spermatozoa. The motility of nanoselected spermatozoa was improved (P < 0.05). The viability of boar sperm, as assessed by the abundance of reactive oxygen species and the integrity of the acrosome, plasma membrane, and mitochondrial membrane was not different between nanoselected and control spermatozoa. The fertility of gilts inseminated with control or nanoselected spermatozoa, as well as growth and health of their offspring were not different between (P > 0.05).ConclusionsThe findings revealed the benefit of magnetic nanoselection for high-throughput targeting of damaged sperm, for removal and rapid and effortless enrichment of semen doses with highly motile, viable, and fertile spermatozoa. Therefore, magnetic nanoselection for removal of abnormal spermatozoa from semen is a promising tool for improving fertility of males, particularly during periods, such as heat stress during the summer months.

157 Dietary L-arginine supplementation affects boar seminal plasma proteome

There are numerous benefit claims for the supplementation of l-arginine in animal production systems. Its positive effect on fertility has been widely characterised in females of various species; however, the effects on the male reproductive system remain debatable with still unfolding molecular mechanisms. Here we investigated the effects of a dietary l-arginine supplementation on boar semen production outcomes, sperm characteristics, and seminal plasma proteome. Nine mature (20 months of age) Nebraska Index Line boars were individually housed and randomly assigned to a soybean meal-based diet containing 0.77% standard l-arginine (control, n=4) or 1.77% high l-arginine (n=5). Boar semen was collected weekly during the 6-week dietary arginine trial. Semen production outcomes (volume and total spermatozoa) and sperm motion (total and progressive motility) and morphology characteristics were subsequently analysed using a computer-assisted sperm analyzer (CEROS II). On Week 6, the seminal plasma of each boar was obtained by centrifugation at 4°C for proteome analysis. The clarified seminal plasma was precipitated; the purified proteins were resuspended in an appropriate buffer and loaded (300μg) onto an immobilized pH gradient strip (isoelectric point 3-10) for the first-dimension electrophoresis. The second-dimension was subsequently performed (4-20% gradient gel), and all gels were stained with Coomassie R250 dye. The PDQuest software (Bio-Rad, Hercules, CA, USA) was used to detect all significantly differentially expressed protein spots (Student’s t-test with P&amp;lt;0.05). Two-way ANOVA repeated measurements (SPSS Statistics, Chicago, IL, USA) was used to analyse dependent (semen outputs and sperm characteristics) and independent (week) variables. A difference was significant at P&amp;lt;0.05. During the l-arginine-consumption trial, semen volumes and total spermatozoa were not significantly affected (P&amp;gt;0.05). Similarly, various sperm motility (total and progressive motility) and velocity characteristics were not affected (P&amp;gt;0.05). By the end of the feed trial, the average (mean±standard error of the mean) semen volume (389±40mL), the total spermatozoa per ejaculate (39±8×109), the total (82±2%) and progressive (62±3%) sperm motility, the sperm velocity (e.g. average path: 98±7μm s−1), and sperm morphology (e.g. distal droplet: 5±1%) parameters of the control group were not significantly different from the group supplemented with high l-arginine: 393±35mL, 47±4×109, 82±2%, 61±3%, 92±8μm s−1, and 5±0%, respectively (P&amp;gt;0.05). In contrast, the proteome profiles of seminal plasma harvested on the sixth week of diet revealed various changes, characterised by the significantly increased expression of 8 protein spots in seminal plasma samples derived from arginine-fed boars (P&amp;lt;0.05). These results indicate that dietary supplementation of l-arginine does not affect the semen outputs, neither the sperm motility characteristics nor their morphology. However, the proteome profile of the seminal plasma was changed by the presence of l-arginine. The findings may have significant implications for boar fertility, and the identification of these proteins is ongoing. This work was supported by USDA-Agricultural Research Service Biophotonics Initiative #58-6402-3-018.

Optimal methods to fix fish sperm for optical microscopic observation: comparisons among different fixative solutions using sperms of copulatory and non-copulatory marine fishes

Sperm fixation in better conditions is a requisite for the examination of sperm morphology using optical microscopes. Here, we investigated the effects of different fixatives on sperm morphological characteristics in four marine fishes: copulatory sculpins Pseudoblennius marmoratus and Radulinopsis taranetzi, and non-copulatory sculpin Icelus mororanis and dragonet Repomucenus beniteguri. We found that a 2.5% glutaraldehyde solution is optimal for observing sperm morphology in these fishes. Furthermore, the low concentrations (2.5%) of formalin could be useful, but the solvent for diluting formalin should be changed depending on the species: seawater in copulatory and non-copulatory sculpins and isotonic solution in the non-copulatory dragonet.

Seminal plasma cell-free mitochondrial DNA copy number is associated with human semen quality

ObjectivePrevious studies have shown that mitochondrial DNA (mtDNA) copy number in human sperm is associated with semen quality. This study examines whether there is association between seminal cell-free mtDNA copy number and human semen parameters. Study designSeminal cell-free mtDNA copy number was measured in 205 men. Semen reactive oxygen species (ROS) level was also measured. Sperm quality was assessed using World Health Organization criteria, while sperm motility parameters were determined by computer-aided sperm analysis. ResultsComparing with normozoospermia, patients with asthenozoospermia and oligoasthenozoospermia had a significant decrease in cell-free mtDNA copy number and an increase in ROS level in seminal plasma (P < 0.05 for all). Seminal cell-free mtDNA copy number positively correlated with sperm concentration, motility, morphology and motion characteristics (r for 0.247 to 0.673, P < 0.05 for all). Semen ROS level negatively correlated with sperm concentration, motility, and motion characteristics (r for −0.261 to −0.676, P < 0.05 for all). There is a negative relationship between seminal cell-free mtDNA copy number and ROS level (r= -0.573, P < 0.05). ConclusionThe results suggested that seminal cell-free mtDNA copy number is associated with semen parameters, which may serve as a novel diagnostic marker of semen quality.

Dependence of Sperm Morphology and Ejaculate Characteristics on Sperm Concentration in the Ejaculates of Hypor Boars.

IntroductionDetermination of sperm concentration and morphology in ejaculate is essential in evaluating fertility. Sperm shape and dimensions may depend on their concentration in ejaculate. The aim of the study was to evaluate the dependence of Hypor boar morphological and morphometric sperm characteristics on concentration in ejaculate.Material and MethodsThe study was conducted on 120 ejaculates from 12 Hypor boars. Depending on sperm concentration, they were put into three groups: low, medium, and high. Ejaculate sperm concentration was determined with the photometric method. Slides were prepared from semen samples which were evaluated for the morphology of spermatozoa. The preparations for morphological analyses were by the eosin-gentian staining method.ResultsAs the sperm concentration in the ejaculate increased, the length, perimeter, and area of the sperm heads also did. In the ejaculates with higher sperm concentration the sperm heads were rounder. The ratios of head dimensions to tail length or total length were the highest in the sperm from ejaculates with the highest concentrations. The highest percentage of morphologically abnormal sperm was noted in ejaculates with low concentrations.ConclusionThe dimensions and shape of sperm depend on the sperm concentration in the ejaculate. In ejaculates with the highest concentrations, the sperm have larger dimensions. Sperm concentration affects the frequency of morphological anomalies in the spermatozoa.