Acrosomal Cap Research Articles

Protective Effect of Aloe vera (L.) on Diabetes-Induced Oxidative Stress Linked Spermiological Co-Morbidity in Human and Rat: An In-Vitro Analysis.

Diabetes linked reproductive complications are rising problems nowadays. The study focused on the protective efficacy of Aloe vera (L.) on sperm cell damage in an oxidative stress milieu encumbered by a chronic diabetes in human and streptozotocin treated Wistar rat (Rattus norvegicus). Spermatozoa from rat's epididymal washing, and human semen after 3-4days of abstinence of mating or masturbation were collected from control and diabetes groups. Spermatozoa of human and rat were incubated for 1 or 2h at 370C in an in-vitro medium separately and considered as normo-glycemic control and diabetes sub-groups. Dose of 1 or, 2 or, 4mg/ml of Aloe vera (L.) hydro-ethanolic (40:60) extract (AVHE) was given to diabetes samples, considered as sub-sub-group for assessing its protective effect on spermiological and oxidative stress parameters. The motility, viability, plasma membrane integrity, nuclear chromatin decondensation for DNA fragmentation, acrosome cap status, and antioxidative status of human and rat spermatozoa were decreased whereas spermatozoal apoptosis was elevated significantly (p < 0.05), noted by TUNEL assay in diabetes samples compared to the duration-matched control group. Exposure of AVHE to diabetes samples resulted significant rectification (p < 0.05) in the said parameters than the unexposed diabetes group. In control group, AVHE exposure has significant protective effect from spermiological deterioration compared to unexposed control group. Identification of major phytomolecules in AVHE was done by LC-MS study. Diabetes-induced oxidative stress-mediated spermatozoal injuries can be protected by AVHE extract, raise the possibility for potentiating sperm of human for increasing the success rate of in-vitro fertilization-blastocyst implantation.

The Effect of Incubation Time on the Quality of Post-Thawed Ram Sexed Sperm

&lt;p&gt;&lt;strong&gt;Objective: &lt;/strong&gt;The sperm sexing using an albumin column requires an incubation process at a certain temperature for sperm to move through the albumin layer. It is thought that the incubation time can influence the quality of the semen resulting from post-thawing sexing. The aim of this research was to determine the effect of incubation time on the quality of sexed sperm post-thawing and to find out the optimum incubation time that provide the best sperm quality.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods:&lt;/strong&gt; This research was carried out in a completely randomized design, using three incubation times (T1= 45 minutes, T2= 60 minutes, T3= 75 minutes) with six replications. The parameters consisted of motility, abnormality and acrosome integrity of sexed sperm in the upper and bottom fractions. Data were analyzed using analysis of variance (Anova) and continued with the Duncan test.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results:&lt;/strong&gt; The results showed that the incubation time had a significant effect on motility, intact acrosome cap and recovery rate, but had no significant effect on abnormality of post thawed sexed sperm. For both the upper and bottom fraction, incubation times of 45 and 60 minutes produced the best motility and recovery rate compared to 75 minutes, but the best intact acrosome cup was obtained from an incubation time of 45 minutes.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; Based on the research results, it can be concluded that the length of incubation time influences the quality of post thawed ram sexed sperm, and 45 minutes is the optimal incubation time to provide the best quality.&lt;/p&gt;

Tex46 knockout male mice are sterile secondary to sperm head malformations and failure to penetrate through the zona pellucida.

Each year, infertility affects 15% of couples worldwide, with 50% of cases attributed to men. It is assumed that sperm head shape is important for sperm-zona pellucida (ZP) penetration but research has yet to elucidate why. We generated testis expressed 46 (Tex46) knockout mice to investigate the essential roles of TEX46 in mammalian reproduction. We used RT-PCR to demonstrate that Tex46 was expressed exclusively in the male reproductive tract in mice and humans. We created Tex46-/- mice using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and analyzed their fertility. Tex46 null spermatozoa underwent further evaluation using computer-assisted sperm analysis, light microscopy, and ultrastructural microscopy. We used immunoblot analysis to elucidate relationships between TEX46 and other acrosome biogenesis-related proteins. Mouse and human TEX46 are testis-enriched and encode a transmembrane protein which is conserved from amphibians to mammals. Loss of the mouse TEX46 protein causes male sterility primarily due to abnormal sperm head formation and secondary effects on sperm motility. Tex46 null spermatozoa morphologically lack the typical hooked sperm head appearance and fail to penetrate through the ZP. Electron microscopy of the testicular germ cells reveals malformation of the acrosomal cap, with misshapen sperm head tips and the appearance of a gap between the acrosome head and the nucleus. TEX46 is essential for sperm head formation, sperm penetration through the ZP, and male fertility in mice, and is a putative contraceptive target in men.

Existence and importance of Na+K+-ATPase in the plasma membrane of boar spermatozoa.

Sodium-potassium-ATPase (Na+K+-ATPase), a target to treat congestive heart failure, is the only known receptor for cardiac glycosides implicated in intracellular signaling and additionally functions enzymatically in ion transport. Spermatozoa need transmembrane ion transport and signaling to fertilize, and Na+K+-ATPase is identified here for the first time in boar spermatozoa. Head plasma membrane (HPM) isolated from boar spermatozoa was confirmed pure by marker enzymes acid and alkaline phosphatase (218±23% and 245±38% enrichment, respectively, versus whole spermatozoa). Western immunoblotting detected α and β subunits (isoforms α1, α3, β1, β2, and β3) in different concentrations in whole spermatozoa and HPM. Immunofluorescence of intact sperm only detected α3 on the post-equatorial exterior membrane; methanol-permeabilized sperm also had α3 post-equatorially and other isoforms on the acrosomal ridge and cap. Mass spectrometry confirmed the presence of all isoforms in HPM. Incubating boar sperm in capacitating media to induce the physiological changes preceding fertilization significantly increased the percentage of capacitated sperm compared to 0h control (33.0±2.6%vs. 19.2±2.6% capacitated sperm, respectively; p=0.014) and altered the β2 immunofluorescence pattern. These results demonstrate the presence of Na+K+-ATPase in boar sperm HPM and that it changes during capacitation.

The effectiveness of Adding Different Sucrose in Tris-Egg Yolk Diluent on the Sperm Fertility of Sexing Swamp Buffalo (Bubalus bubalis)

Spermatozoa sexing requires a diluent that can protect and provide an optimal environment to maintain the quality of spermatozoa. This study targets to determine the effectiveness of adding sucrose to tris-egg yolk diluent on the sperm quality of the swamp buffaloes produced by sexing with the egg albumin method. Semen was collected once a week for 12 consecutive weeks with an artificial vagina from three male buffaloes at the Indonesian Center for Animal Research and Development (ICARD) in April - June 2021. Semen was separated by sedimentation using 10% and 30% egg albumins. Semen from sexing was divided into four diluent treatments, namely 20% tris-egg yolk (TEY), TEY+0% sucrose, TEY+0.2% sucrose, TEY+0.3% sucrose, and TEY+0.4% sucrose. Parameters measured included: motility, viability, intact plasm membrane, and intact acrosome cap of buffalo sperm. The results showed the motility of X sperm: 43.75-47.5%, Y sperm: 40-45%, the viability of X sperm: 78.25-79.75% and Y sperm: 77.25-79.75%, intact plasm membrane of X sperm: 70.5-71.5% and Y sperm: 70.5-71%, and viable acrosome-intact for X sperm: 79-80% and Y sperm: 78.75-79.5%. The addition of sucrose up to 0.4% into tris-egg yolk diluent was proven can maintain motility, viability, and integrity of cell membrane and acrosome of spermatozoa of water buffalo that was sexed with egg albumin.

Influence of Sperm Number and Antioxidant Melatonin in Extender on the Quality of Post-Thawing Sheep Spermatozoa

This study aimed to examine the effect of spermatozoa concentration and the effectiveness of melatonin supplementation in diluent on the quality of post-thawing semen. Ejaculated semen was collected using the artificial vaginal method (MVB). The study was carried out in two stages, firstly semen was frozen in andromed diluent with different concentrations in one straw (50, 100, 200 million per straw), and the second was frozen semen in diluent supplemented with melatonin with different doses (0, 0,5, 1, 0 and 1.5 mM melatonin). Parameters observed were the movement of spermatozoa using Computer Assisted Sperm Analysis, membrane integrity, and acrosome integrity. Data were analyzed using ANOVA and further tested using Duncan's test. The results showed no significant difference in the quality and movement pattern of sheep semen when frozen at concentrations of 50, 100, or 200 million per straw (P 0.05). Furthermore, adding melatonin to the diluent in this study affected spermatozoa's total motility and progressive motility at a concentration of 1.0 mM (P0.05) but did not significantly affect the percentage of spermatozoa motility pattern characteristics. From the results, it can be concluded that the concentration of per straw spermatozoa does not affect the quality of sheep semen, and melatonin supplementation in diluent can reduce the effects of the frozen-thawed process on motility, acrosome cap, and plasma membrane integrity in sheep semen. Melatonin supplementation with a concentration of 1 mM in the extender was the highest quality concentration in this study.

Deterioration of Frozen Semen of Bali Cattle after Cooling at 5°C

Frozen semen is produced through several stages, which deteriorate spermatozoa. This research aimed to evaluate the deterioration degree of frozen semen after 5 °C cooling and freezing of Bali cattle. The samples included 10 male Bali cattle with a body weight of 542-668 kg, from which semen was collected once a week for five weeks. The deterioration of each individual’s sperm was determined by observing two distinct straws. The parameters observed included viability, abnormalities, intact plasma membrane, and intact acrosome cap. Initial observations of the parameters were conducted following the addition of semen to diluent A1 (AD) as much as the volume of fresh semen. The semen in the AD group was not cooled and frozen. The A1 semen was then divided into two, namely, those with cooling at 5 °C for 4 hours (PT1) and at 5°C for 22 hours (PT2). The results showed that individual variations in Bali cattle caused significant differences in viability and intact plasma membrane of AD and PT1 groups, while PT2 did not differ in viability and intact plasma membrane spermatozoa. Abnormalities were significantly different between AD and PT2 groups, however PT1 did not differ in abnormalities spermatozoa. Intact acrosomal cap was significantly different in the AD, PT1, and PT2 groups. In conclusion, individual variations, including viability, abnormalities, intact plasma membrane, and acrosome cap of spermatozoa, were better at 4 hours compared to cooling at 5°C for 22 hours. A Cooling time of 4 hours at 5°C can be recommended for frozen semen processing.

A comprehensive investigation of human endogenous retroviral syncytin proteins and their receptors in men with normozoospermia and impaired semen quality.

The study aims to investigate first the presence of Syncytin 2 and its receptor, MFSD2, in human sperm, and second whether the expressions of Syncytin 1, Syncytin 2, and their receptors, SLC1A5 and MFSD2, differ between normozoospermic, asthenozoospermic, oligozoospermic, and oligoasthenozoospermic human sperm samples. The localization patterns and expression levels of syncytins and their receptors were evaluated in normozoospermic (concentration = 88.9 ± 5.5 × 106, motility = 79.2 ± 3.15%, n = 30), asthenozoospermic (concentration = 51.7 ± 7.18 × 106, motility = 24.0 ± 3.12%, n = 15), mild oligozoospermic (concentration = 13.5 ± 2.17 × 106, motility = 72.1 ± 6.5%, n = 15), moderate oligozoospermic (concentration = 8.4 ± 3.21 × 106, motility = 65.1 ± 8.9%, n = 15), severe oligozoospermic (concentration = 2.1 ± 1.01 × 106, motility = 67.5 ± 3.2%, n = 15), and oligoasthenozoospermic (concentration = 5.5 ± 3.21 × 106, motility = 18.5 ± 1.2%, n = 15) samples by immunofluorescence staining and western blot. Syncytins and their receptors visualized by immunofluorescence showed similar staining patterns with slight staining of the tail in all spermatozoa regardless of normozoospermia, asthenozoospermia, oligozoospermia, or oligoasthenozoospermia. The localization patterns were categorized as equatorial segment, midpiece region, acrosome, and post-acrosomal areas. The combined staining patterns were also detected as acrosomal cap plus post acrosomal region, the midpiece plus equatorial segment, and midpiece plus acrosomal region. However, some sperm cells were categorized as non-stained. Both syncytin proteins were most intensely localized in the midpiece region, while their receptors were predominantly present in the midpiece plus acrosomal region. Conspicuously, syncytins and their receptors showed decreased expression in asthenozospermic, oligozoospermic, and oligoasthenozoospermic samples compared to normozoospermic samples. The expression patterns of HERV-derived syncytins and their receptors were identical regardless of the spermatozoa in men with normozoospermia versus impaired semen quality. Further, asthenozoospermia, oligozoospermia, and oligoasthenozoospermia as male fertility issues are associated with decreased expression of both syncytins and their receptors.

PDCL2 is essential for sperm acrosome formation and male fertility in mice.

Each year, infertility affects 15% of couples worldwide, with 50% of cases attributed to men. Globozoospermia is an uncommon cause of male factor infertility, characterized by defects in sperm acrosome formation, leading to round-headed spermatozoa. We generated Pdcl2 knockout mice to investigate the essential roles of PDCL2 in mammalian reproduction. We used reverse transcription-polymerase chain reaction to demonstrate that PDCL2 was expressed exclusively in the male reproductive tract in mice and humans. We created Pdcl2 knockout mice using the CRISPR-Cas9 system and analyzed their fertility. Pdcl2 null spermatozoa underwent further evaluation using computer-assisted sperm analysis, light microscopy, and ultrastructural microscopy. We used immunoblot analysis and immunofluorescence to elucidate relationships between PDCL2 and other acrosomal proteins. The PDC family is highly conserved in eukaryotes. Mouse and human PDCL2 are testis enriched and localized to the testicular endoplasmic reticulum. Loss of the protein causes sterility because of abnormal acrosome biogenesis during spermiogenesis and immotility. Furthermore, Pdcl2 null spermatozoa have rounded heads, similar to globozoospermia in humans. Observation of the knockout testis shows a lack of acrosomal cap formation, aberrant localization of mitochondria in the sperm head, and misshapen nuclei. PDCL2 is essential for sperm acrosome development and male fertility in mice and is a putative contraceptive target in men.

Daya Tahan Tudung Akrosom dan Membran Plasma Spermatozoa Sapi Bali yang Dipaparkan pada Suhu Ruang

This study aimed to determine the effect of storage at room temperature on the durability of the acrosome cap and plasma membrane of Bali cattle spermatozoa. This research was conducted at the Reproduction Laboratory of the Faculty of Animal Husbandry, Jambi University. The experiment was carried out using a completely randomized design with 4 treatments and 6 replications. Storage treatment at room temperature consisted of T0: no storage (control),T1: storage for 2 hours, T2: storage for 4 hours, T3: storage for 6 hours. The data obtained from each variable analyzed with variance. The treatment had a significant effect, so Duncan's further test was carried out. The process of calculating the data using the SPSS program. From the results of this study, it was found that storage of spermatozoa at room temperature reduced the quality of Bali cattle's spermatozoa against the acrosome hood, plasma membrane, and spermatozoa recovery rate (P&lt;0.05). At storage for up to 4 hours, both 44,33% acrosomal cap and 52.83% recovery rate were still eligible for insemination. In plasma membrane storage at room temperature ranging from 0 hours 56.60% to 6 hours 35.83% showed a fairly low average, which was below 60% as the minimum requirement for insemination

Effectiveness of Various Glycerol Concentrations as a Cryoprotectant in Frozen Semen of Pasundan Cattle

This study was conducted to determine the effectiveness of Glycerol as a cryoprotectant with various concentrations on the quality of Pasundan cattle semen. Semen was collected from seven bulls of Pasundan Cow using an artificial vagina twice a week for three months. The semen sample was added with a TRIS-Egg Yolk Extender containing 20% (v/v) egg yolk and treated with the addition of Glycerol with five different concentrations (G5=5%, G6=6%, G7=7%, G8= 8%, and G9=9%) were then performed with cryopreservation. A Completely Randomized Design (CRD) was used to examine the effect of five different concentrations of Glycerol on motility, intact plasma membrane (IPM), the integrity of acrosome cap (IAC), abnormalities, and recovery rate (RR) of spermatozoa after cryopreservation (post-thawing). The results of diluted Pasundan cattle semen evaluation showed that the addition of 7% Glycerol (G7) resulted in the best motility and IAC values (83.68% and 72.84%), the addition of 7% and 8% Glycerol (G7 and G8) resulted in the best IPM values (85.00% and 84.50%). The addition of 6%, 7%, 8%, and 9% Glycerol (G6, G7, G8, and G9) resulted in the lowest abnormality values (1%). On the post-thawing Pasundan cattle semen evaluation, the addition of 7% Glycerol (G7) resulted in the best motility, IAC, IPM, and RR values (54.49%, 38.57%, 54.29%, and 72.28%). Meanwhile, adding various Glycerol concentrations did not significantly affect the abnormality value of post-thawing spermatozoa. Generally, the addition of 7% Glycerol in semen extenders shows optimal results as a cryoprotectant.

Optimizing Reproductive Performances of Garut Sheep as Superior Local Genetic Resources by Modification of Dietary Grass and Concentrate Balances

High-quality Garut rams are important to be selected for Garut sheep development to include improving their semen qualities. The semen quality is highly affected by dietary nutrient intake. This research aimed to conclude the effect of different grass and concentrate balances on performance and semen qualities such as motility, abnormality, the integrity of plasma membrane, and acrosome cap sperms of Garut rams. This research was conducted from August 25th, 2020, to February 2nd, 2021, at Margawati Sheep Breeding Center. A Completely Randomized Design (CRD) was used to compare 3 different grass and concentrate balance treatments (T1 = 80% grass: 20% concentrate; T2 = 60% grass: 40% concentrate; and T3 = 40% grass: 60% concentrate) on performance and semen qualities of Garut rams using 6 replicates (n = 6). The data were analyzed using analysis of variance and Duncan's test. The results showed there was no different among the treatments on average daily gain (ADG, g/head/day) and dry matter intake (DMI, g/head/day) but increasing level of dietary concentrate resulted in increased motility (P<0.01), reduced abnormality (P<0.05), increased integrity of plasma membrane (P<0.001) and increased acrosome cap (P<0.001) of sperms of Garut Rams. The T2 treatment resulted in an optimal grass (60%) and concentrate (40%) balance. In conclusion, increased dietary concentrate did not significantly increase the daily weight gain, but it improved the Garut ram’s semen quality.

Characteristics of Bali Bull (Bos Sondaicus) Sexed Sperms with Freeze Dry Egg White at Different Incubation Time

The development of reproductive technology for the separation of spermatozoa with X and Y chromosomes, which is better known as sexing, can be carried out in vitro using various methods, one of which is using freeze dry egg whites. This study aims to determine the quality of semen based on incubation time. This study used four types of treatment, namely P0 = No Incubation, P1 = Incubation 30 minutes, P2 = Incubation 45 minutes, P3 = Incubation 60 minutes with six replications. Parameters observed were the quality of spermatozoa macroscopically (volume, odor, color and consistency) and microscopically (motility, abnormality, concentration, intact plasma membrane, intact acrosome cap). Y using descriptive statistical test. The results of analysis of variance that sexing spermatozoa using freeze dry egg whites at different incubation times showed a significant effect (P&lt;0.05) on intact plasma membrane (IPM), intact acrosomal membrane (IAM), but had no significant effect P&gt;0.05) on concentration, motility, and abnormalities. Evaluation of fresh Bali cattle spermatozoa showed results that were in accordance with the standards for further examination of semen sexing. Incubation time of 30 minutes in the sexing process of spermatozoa caused a significant decrease in motility and concentration, meanwhile, IPM and IAM of sexed spermatozoa experienced a significant decrease in incubation time of 60 minutes, but the decrease was still within the normal range for spermatozoa. to be used in the artificial insemination process. Keywords: Sexing, incubation, concentration, motility, abnormality, intact plasma membrane, intact acrosome caps.

Spatial Expression of Osteopontin in Testis, Epididymis and Spermatozoa in Dromedary Camel

The aim of this study is to clarify the function of osteopontin (OPN) through its expression and localisation in male testis, epididymis and sperm cell of dromedary camel during rutting season. Testis and epididymis parts (caput, corpus and cauda) specimens were obtained from 8 mature male camels and semen was collected from 4 another fertile camels for sperms. OPN mRNA expression and its location was analysed using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) techniques, respectively. OPN mRNA expression was significantly (P < 0.05) higher in the cauda of epididymis than other epididymal parts and testis. Immunolocalisation revealed high immunoreaction intensity in the elongated spermatids in seminiferous tubules and Leydig cells in interstitial tissue. However, a faint immunostaining was seen in spermatogonia and Sertoli cells. Along the epididymis tract, intense immunoreactivity of OPN was localised in the cytoplasm of the epithelial cells with high concentration in the apical region nearby lumen and the acrosomal part of the cauda epididymal sperms. Immunofluorescence and immunochromogenic staining of OPN was detected on the acrosomal cap and neck of epididymal sperms. These findings suggested that OPN might play an important role in the sperm protection, migration and fertilisation in dromedary camels.

닭 신선 정자 운동성과 첨체안정성에 보존 온도, 희석제 및 용기가 미치는 영향

Chicken spermatozoa have the ability to survive in low-temperature environments; however, the effects of low temperature on sperm motility and acrosome damage have not been studied in detail. The present study investigated semen longevity following dilution of rooster semen with Beltsville Poultry Semen Extender (BPSE) and Lake extender in preservation vessels (1.5 mL e-tube and 0.5 mL straw). Spermatozoa motility in the closed-type vessel (0.5 mL straw) was higher than that in the 1.5 mL e-tube on day 3 of preservation (68.6±3.1% vs. 22.1±5.7%). The motility of rooster semen diluted with BPSE in 0.5 mL straw was also higher than that of the Lake extender on day 3 of preservation (57.7±5.6% vs. 37.7±5.4%). Furthermore, acrosome intactness was higher in 0.5 mL straw than in the 1.5 mL e-tube, and the rate of acrosome cap damage increased with preservation days. The present study demonstrates that a closed 0.5-mL straw vessel could be used for low-temperature semen preservation, with an increased motility rate and acrosome integrity in fresh rooster semen.

Sperm acrosome status before and during fertilization in the Chinese hamster (Cricetulus griseus), and observation of oviductal vesicles and globules.

The present study was conducted to determine exact location where the acrosome reaction of fertilizing spermatozoa begins in the oviduct of the Chinese hamster. Unlike spermatozoa of other rodent species, Chinese hamster spermatozoa did not spontaneously undergo the acrosome reaction in fertilization-supporting media. In naturally mated females, spermatozoa in the uterus had intact acrosomes, whereas those in the lower oviductal isthmus had visibly thin acrosomal caps. The acrosomal cap was lost when spermatozoa passed through the cumulus oophorus. Thus, Chinese hamster spermatozoa begin the acrosome reaction in the lower isthmus and complete it in the cumulus oophorus. The mucosal epithelium of the oviductal isthmus released many "transparent" vesicles into the lumen, was very fragile and readily sloughed off by rough handling or rapid flushing with medium. Globular materials that oozed out of the dissected oviduct were most likely mucosa cells destroyed by rough handling. Although the oviducts of Chinese hamsters may be exceptionally delicate, this observation nevertheless warns us to cautiously handle the oviducts of any species when studying oviduct secretions that could be involved in inducing capacitation and the acrosome reaction of spermatozoa within the female genital tract.

The quality of Etawah crossbreed sperm after sexing with different combination of Bovine Serum Albumin concentrations

This research aimed to determine the quality of Etawah crossbreed sperm after sexing with different combinantion of Bovine Serum Albumin (BSA) concentrations. The parameter of this research were motility, viability, intact plasma membrane (IPM) and intact acrosome cap (IAC) (%). The Completely randomized design (CRD) was applied in this experiment involving 4 treatments of the combination of BSA concentrations (T1=3%:6%, T2 = 4%:8%, T3=5%:10%, T4=6%:12% at upper and lower fraction) and each treatments was repeated 5 times at post chilled and post sexing. Data were analyzed by using analysis of variance (ANOVA), followed by Duncan Multiple Range Test. The result showed that thecombination of BSA concentrations affected (P<0.05) motility, viability, IPM and IAC. The highest value of sperm quality in upper and lower fraction was obtained from the combination BSA of 5%:10% (motility 78.60 ± 2.61% and 73.80 ± 2.49%; viability 282 ± 14.30 and 252.8 ± 12.97 hours; IPM-value 79.60 ± 1.98% and 74.70 ± 1.82% and IAC 81.00 ± 1.46% and 76.90 ± 1.29%). Based on the results it can be concluded that the quality of Etawah crossbreed sperm after sexing is affected by the combination of BSA concentrations.

αV Integrin Expression and Localization in Male Germ Cells.

Integrins are transmembrane receptors that facilitate cell adhesion and cell–extracellular matrix communication. They are involved in the sperm maturation including capacitation and gamete interaction, resulting in successful fertilization. αV integrin belongs to the integrin glycoprotein superfamily, and it is indispensable for physiological spermiogenesis and testosterone production. We targeted the gene and protein expression of the αV integrin subunit and described its membrane localization in sperm. Firstly, in mouse, we traced αV integrin gene expression during spermatogenesis in testicular fraction separated by elutriation, and we detected gene activity in spermatogonia, spermatocytes, and round spermatids. Secondly, we specified αV integrin membrane localization in acrosome-intact and acrosome-reacted sperm and compared its pattern between mouse, pig, and human. Using immunodetection and structured illumination microscopy (SIM), the αV integrin localization was confined to the plasma membrane covering the acrosomal cap area and also to the inner acrosomal membrane of acrosome-intact sperm of all selected species. During the acrosome reaction, which was induced on capacitated sperm, the αV integrin relocated and was detected over the whole sperm head. Knowledge of the integrin pattern in mature sperm prepares the ground for further investigation into the pathologies and related fertility issues in human medicine and veterinary science.

Seminal plasma Alters surface Glycoprofile of dromedary camel cryopreserved epididymal spermatozoa

The high viscosity of Camelidae semen continues to present a major impediment for its application in assisted reproduction technology. The exposure of epididymal spermatozoa (ES) to seminal plasma (SP) may provide an approach to enhance the development of assisted reproductive techniques in these important domestic species. Since the sperm glycocalyx plays a key role in reproduction we aimed to evaluate whether SP exposure modifies the surface glycosylation patterns of cryopreserved dromedary ES. Epididymal sperm was collected through retrograde flushing of the cauda epididymidis that were obtained from orchidectomized mature dromedary bulls. The collected samples were then cryopreserved after dilution with a tris citrate clarified egg yolk extender, with and without the supplementation of 15% SP. Post-thaw carbohydrate surface profiles of both control and SP-treated spermatozoa were analyzed using 15 fluorescent lectins. Morpho-functional properties were also investigated via computer assisted sperm analysis. Lectin-binding analysis of the glycocalyx in control sperm revealed the presence of (1) N-glycans terminating with lactosamine (Con A, PHA-L, and RCA120), in both acrosomal and tail regions. Whilst (2) α2,3-/α2,6-linked sialic acids (MALII, SNA), and O-linked glycans terminating with a single N-acetylgalactosamine residue (Tn antigen) (HPA, SBA) along with galactoseβ1,3N-acetylgalactosamine (T antigen) (PNA) were observed in the acrosomal cap. The expression of both N-acetylglucosamine (sWGA and GSA II) and terminalαgalactose (GSA I–B4) residues was also noted in the acrosomal cap region of control sperm. Compared with controls, SP treated samples displayed: 1) the appearance of bisected di-triantennary complex-type N-glycans (PHA-E), terminating with lactosamine, as well as an increase of O-glycans terminating with Tn and T antigens in both the acrosomal and tail regions; 2) an increase in glycans containing α2,6-linked sialic acid, N-acetylglucosamine, and αgalactose in the tail region. The cytoplasmic droplets of both control and seminal plasma-treated sperm bound Con A, PHA-E, PHA-L, RCA120, HPA, PNA, sWGA, GSA I–B4, and GSA II. These results indicate that SP treatment affects the glycan composition of the dromedary camel ES glycocalyx. More comprehensive studies are required in order to evaluate the fertilization capacity of SP-treated ES in order to facilitate its application in dromedary camel assisted reproduction technology.

Localization and expression of ADAM2 in the dromedary camel testis, epididymis and sperm during rutting season.

ADAM2 (fertilin β) is a sperm surface protein reported in several mammalian species. However, the presence of ADAM2 in the male reproductive system and sperm of the camel is not well known. The present study was to clarify the localization and expression of ADAM2 in the dromedary camel testis, epididymis and spermatozoa during rutting season using immunohistochemistry (IHC) and the quantitative real-time polymerase chain reaction (qPCR). Tissue samples were obtained from the testis (proximal and distal) and epididymis (caput, corpus, and cauda) from eight mature male camels. Epididymal and ejaculated sperms were collected from four other fertile camels. IHC analysis clearly showed the localization of ADAM2 protein in the spermatocytes and the round and elongated spermatids of the testis, in the epithelial cells along the epididymis tract, on the posterior head of the sperm within the cauda epididymis, and on the acrosomal cap of both the epididymal and ejaculated sperm. The expression of camel ADAM2 mRNA was significantly higher (P < 0.05) in the testis when compared with the epididymis. These findings may suggest an important role of ADAM2 in the fertility of male dromedary camels.