Intact Sperm Research Articles

In vitro Supplementation of Linoleic Acid Improves Quality of Cryopreserved Buffalo Semen

Present study was designed to evaluate the effect of linoleic acid (LA) supplementation in extender on post thaw quality of cryopreserved buffalo semen. Semen was collected from three adult Nili Ravi buffalo bulls of same age with artificial vagina (42°C) for five weeks (replicates; N=30). Qualified semen ejaculates (>1mL volume, >60% motility, >0.5 billion/mL concentration) were diluted in tris-citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0ng mL-1 of LA and were cryopreserved using standard procedures. Sperm motility and plasma membrane integrity were improved plessthan0.05) in extender containing 10.0 ng mL-1 of LA compared to other treatments and control while number of acrosome intact live sperm, chromatin integrity and number of morphologically normal sperms remained the same. In conclusion, LA supplementation in extender at 10.0 ng mL-1 was found to be beneficial to improve post thaw quality of cryopreserved buffalo semen.

Relationship of sperm plasma membrane and acrosomal integrities with sperm morphometry in Bos taurus.

To date, sperm morphometric studies have assessed whole sperm populations without considering sperm function. The aim of this study was to evaluate the relationship of sperm membrane and acrosomal integrity with sperm morphometry in liquid semen samples collected from bulls. To this end, sperm morphometry was performed on cryopreserved semen samples from 16 bulls by a combination of fluorescent dyes, including Hoechst 33343, carboxyfluorescein diacetate, and propidium iodide. This allowed discrimination of different subpopulations on the basis of sperm membrane and acrosomal integrity and analysis of the morphometrics of the sperm head, nucleus, and acrosome using a specific plug-in module created on ImageJ. Acrosomal integrity was related to sperm morphometry as the heads of spermatozoa with a damaged acrosome were significantly smaller than those with a normal acrosome (P < 0.001). In the case of spermatozoa with an intact acrosome, those with a damaged plasma membrane had a larger sperm head and acrosome than spermatozoa with an intact plasma membrane (P < 0.001). No significant differences in the sperm head size were observed between sperm subpopulations without an acrosome or in the nuclear sperm morphometry of the different subpopulations. There was a positive correlation between the sperm motility values of the samples and the morphometric parameters for intact spermatozoa. These correlations were particularly strong for the morphometric parameters of the sperm acrosome. We conclude that there are clear differences in the sperm morphometry depending on the status of the sperm membrane and acrosome and this should be considered when performing this kind of analysis.

The effect of crude guava leaf tannins on motility, viability, and intact plasma membrane of stored spermatozoa of Etawa crossbred goats.

Aim:The aim of this study was to know crude guava leaf tannins effect on motility, viability, and intact plasma membrane of stored spermatozoa of Etawa crossbred goats.Materials and Methods:Macroscopic assessment of normal Etawa crossbred semen was followed by dilution with a glucose solution at a 1:10 ratio to increase volume. The diluted semen was treated by adding crude guava leaf tannins into 1 ml of the semen glucose diluent, and five treatments were obtained, namely, control group (C), with no added tannins; treatment Group 1 (T1), with 3%; treatment Group 2 (T2), with 6% tannins; treatment Group 3 (T3), with 12% tannins; and treatment Group 4 (T4), with 24% tannins. Each treatment used five replications. Then, microscopic analysis of the treated and control semen was carried out after 15 days of storage at 4-5°C temperature. The parameters observed were motility, pH, viability, abnormality, and intact spermatozoa plasma membrane.Results:The spermatozoa motility in Group C was the highest (76.60±1.47). The motility in Group T1 did not differ from that in Group C, but was different and higher than that in Groups T2, T3, and T4. The pH of Group C tended to be acidic after 15 days of storage (4.78±0.01) as compared to the initial pH of fresh semen (6.76±0.12). The pH in Group C did not differ from that in the Groups T1 and T2, but differed from that in the T3 and T4 groups; the pH in the T3 and T4 groups was similar. The viability of spermatozoa in the T1 group was higher than that in all treatments (64.60±2.76); the lowest values were found in Group C (28.94±1.02). Group C had the lowest number of normal spermatozoa, with a mean of 72.58±3.48. The total number of abnormalities in the T2 group did not differ from those in the T3 group, and abnormalities in the T4 group did not differ from those in Group C, which exhibited the highest abnormalities in the head, neck, and tail. The most significant decrease was observed in the intact plasma membrane of spermatozoa on addition of 12% and 24% crude guava leaf tannin in glucose diluents.Conclusion:The addition of 3% crude guava leaf tannin to crossbred Etawa goat semen diluted with glucose diluent and stored for 15 days at 4-5°C resulted in a significant effect on spermatozoa motility, viability, and intact plasma membrane, whereas the administration of 24% crude guava leaf tannin resulted in low live percentage of spermatozoa.

Effect of chelating and antioxidant agents on morphology and DNA methylation in freeze-drying rabbit (Oryctolagus cuniculus) spermatozoa.

Freeze-drying (FD) has been exhaustively tried in several mammalian species as an alternative technique to sperm cryopreservation, but few studies have been done in rabbits (Oryctolagus cuniculus). The main objective of this study was to compare the protective effect of various antioxidants added to EDTA medium on structural and functional components of FD rabbit spermatozoa and on their status of global DNA methylation. FD media used were composed of basic FD medium (10mM Tris-HCl buffer and 50mM NaCl) supplemented with either 50mM EDTA alone (EDTA) or added with 105µM of rosmarinic acid (RA, EDTA-RA) or 10µM of melatonin (MLT, EDTA-MLT). The effect of each medium on the preservation of FD spermatozoon structure was evaluated with light and scanning electron microscopy (SEM). Global DNA methylation was quantified in all FD sperm samples as well as in fresh spermatozoa. Morphologically, fracture points were evidenced in the neck, mid and principal piece of the spermatozoon tail. No differences in spermatozoon fracture points were evidenced among FD treatments: intact spermatozoa were the largest (p<.01) category, whereas the most frequent (p<.01) injury was the neck fracture, resulting in tailless heads. At SEM, the head of spermatozoa showed a well-conserved shape and intact membrane in all treatments. DNA methylation status was the same in all FD treatments. In conclusion, supplementation of EDTA, EDTA-RA and EDTA-MLT during FD preserved rabbit sperm morphological integrity and methylation status as well. Therefore, the difficulty of getting viable offspring using FD semen is likely unrelated to the impact of the lyophilization process on DNA methylation and morphology of lyophilized spermatozoa.

Effects of TLR4 overexpression on sperm quality, seminal plasma biomarkers, sperm DNA methylation and pregnancy rate in sheep

Genetic modification provides a means to enhancing disease resistance in animals. In this study, the first generation of genetically modified (GM) sheep overexpressing TLR4 was produced by microinjection for better disease resistance. To compare semen characteristics including sperm quality, seminal plasma biochemical index, sperm DNA methylation and pregnancy rate of three-year old transgenic sheep with TLR4 overexpressed (toll like receptor 4, TLR4) and non-transgenic ram. Sixteen transgenic ram of F0 generation were produced by microinjection of the TLR4 plasmid into the pronucleus of fertilized ova. Seven transgenic sheep of F1 generation was produced by breeding F0 transgenic founders with non-transgenic sheep of the same breed. There were no significant differences between transgenic and control rams for all semen quality parameters, including semen volume, sperm concentration, sperm viability, and percentages of sperm with an intact plasma membrane, acrosomal integrity, and viable sperm with high mitochondrial membrane potential in both F0 and F1 generation. Furthermore, no significant differences were found for seminal plasma concentrations of zinc, neutral alpha-glucosidase, acid phosphatase or fructose, nor for levels of H19 and IGF2R methylation in sperm DNA. In addition, pregnancy rate was also similar between these two groups. In conclusion, there was no evidence that TLR4 overexpression altered the sperm quality, seminal plasma or sperm DNA of transgenic sheep.

Silk protein sericin pretreatment enhances osmotic tolerance and post-thaw sperm quality but reduces the ability of sperm cells to undergo in vitro induced acrosome reaction in rabbit

Effect of sericin pretreatment of sperm cells on the osmotic tolerance, ability to undergo acrosome reaction induced by calcium ionophore (CI), heparin (H) or lysophosphatidylcholine (LPC), post-thaw sperm quality and in vivo fertility was evaluated in four successive experiments in rabbit. In experiment 1, fresh semen was pretreated with sericin (0, 0.1% or 0.5% w/v) before exposing to fructose solutions adjusted to either 50, 100, 290, 500 or 1000 mOsm/L. Sericin pretreatment increased sperm livability in addition to live-membrane intact and total membrane intact sperm rates (P < 0.05) in 50 and 290 mOsm/L groups. In experiment 2, sperm samples were pretreated by either 0.1 or 0.5% sericin after removal of the semen plasma. CI, H or LPC were used to induce acrosome reaction in pretreated sperm samples. Sericin pretreatment, reduced the ability of sperm cells to undergo acrosome reaction (P < 0.05) in vitro. In experiment 3, ejaculates were frozen with or without sericin pretreatment in DMSO-sucrose extender. In post-thaw samples sericin pretreatment improved total and progressive motility, livability, membrane and acrosome integrity in a dose dependent manner (P < 0.05). In vivo fertility trials by artificial inseminations revealed contradictory results in experiment 4. Although 0.5% sericin pretreatment totally inhibited fertility, 0.1% sericin provided high pregnancy rates. In conclusion; sericin pretreatment enhances osmotic tolerance and post-thaw sperm quality, but reduces the ability of rabbit sperm cells to undergo in vitro induced acrosome reaction, but this effect is restored in vivo by dose dependent manner.

Characterization of antisperm antibody binding patterns in relation to sperm phenotypic attributes and field fertility in dairy bulls

To test our hypothesis that antisperm antibodies (ASA) might alter sperm phenotypic attributes thus leading to sub-fertility/infertility in bulls, ASA were generated in crossbred male calves by immunizing with sperm two times. Cryopreserved spermatozoa from crossbred bulls (n = 24) with different field fertility ratings were incubated with ASA and different patterns of ASA immunolocalization were studied. In addition, sperm membrane integrity, acrosomal integrity and cryo-capacitation status were also assessed. Immunolocalization of sperm antigens using antisperm antibody revealed three major patterns (Acrosomal-AR, apical-AP and, acrosome and tail-AT). The proportion of ASA reactive spermatozoa was significantly (P < 0.05) higher in low- and medium-fertile bulls compared to high-fertile bulls. Among the three patterns, the proportion of spermatozoa with AR pattern was significantly (P < 0.05) higher in low- and medium-fertile bulls compared to high-fertile bulls. The proportion of membrane and acrosome intact spermatozoa was significantly (P < 0.05) higher in high-fertile bulls compared to medium- and low-fertile bulls. There were no significant differences in the proportion of cryo-capacitated spermatozoa among high-, medium- and low-fertile bulls. The relationship between ASA reactive spermatozoa and conception rates (CR) of bulls was highly (P < 0.01) significant and negative. Similarly, AR and AT pattern were also significantly (P < 0.01) and negatively related to CR of bulls. The reactivity of spermatozoa with ASA was also significantly (P < 0.01) and negatively related to the membrane and acrosome integrity of spermatozoa. It was concluded that the proportion of spermatozoa responding to ASA was higher in low-compared to high-fertile bulls and ASA localization in sperm acrosomal area was negatively related to sperm membrane and acrosomal integrity and bull fertility.

Effect of artificial vagina lubricants on stallion sperm quality

Commercially available vaginal lubricants, typically labeled as non-spermicidal, are used to lubricate equine artificial vaginas prior to semen collection. Improper type or amount of lubricant might affect stallion sperm quality, either after short-time exposure or following cooled storage of extended semen previously exposed to lubricant. The aim of this study was to evaluate stallion sperm quality following exposure to lubricant-containing extender for 1 h (T1h) or 24 h (T24h). Three ejaculates were collected from each of four stallions using a small volume of petrolatum to lubricate artificial vaginas, and gel-free semen was diluted to 30 × 106 sperm/mL in extender containing: no lubricant (control), or 1 or 5% (v/v) HR® Lubricating Jelly (HR1 or HR5); K-Y® Jelly (KY1 or KY5); Therio-gel® (TG1 or TG5); Priority Care® Sterile Lubricating Jelly (PC1 or PC5); or Clarity® A.I. Lubricating Jelly (CL1 or CL5). Sperm were evaluated at T1h and T24h for percentages of: total and progressive sperm motility (TMOT and PMOT); curvilinear velocity (VCL; μm/s); and straightness (STR; %); viable acrosome intact sperm (VAI); sperm with abnormal DNA (COMP-αt); viable lipid peroxidation negative sperm (VLPN); and sperm with no detectable DNA oxidative injury [8OHdG(−)]. Following short-term exposure of sperm to lubricants, KY5 reduced TMOT, PMOT, VCL, VAI, VLPN, and COMP-αt in comparison with controls (i.e., P < 0.05). PC5 reduced TMOT, PMOT, VCL, VAI, and 8OHdG(−), and KY1 reduced TMOT, VAI, VLPN in comparison to controls (P < 0.05). Lubricant CL1, HR1 and HR5 yielded similar values to controls for all 8 endpoints, and CL5 yielded similar values to controls for all 8 endpoints (P > 0.05), except for VCL. Following long-term exposure, KY5 decreased TMOT, PMOT, VCL, VAI, VLPN, and COMP-αt as compared to controls (i.e., P < 0.05), PC5 decreased TMOT, VCL, VAI, and 8OHdG(−)as compared to controls in PC5, and KY1 decreased TMOT, VAI, VLPN, and COMP-αt (P < 0.05). TG5 decreased TMOT, PMOT, and VCL as compared to controls (P < 0.05). Lubricant CL5 decreased VCL (P < 0.05), and CL1, HR5, HR1, PC1, and TG1 were similar to controls for all 8 endpoints (P > 0.05). Overall, lubricant KY was the most detrimental to sperm quality, with most profound changes detected at a 5% concentration. Lubricants CL and HR were generally similar to controls and were less affected by lubricant concentration.

Advanced sperm selection techniques for assisted reproduction.

Advanced sperm selection techniques for assisted reproduction.

Ramifications of protease-based liquefaction of camel semen on physical, kinematic and surface glyco-pattern of cryopreserved spermatozoa

The efficiency of incorporating different proteases in the diluent for reducing camel semen viscosity, and subsequent ramifications on morpho-functional and glycan surface properties of cryopreserved spermatozoa were investigated. Ejaculates (n = 48) were collected from three adult camels, Camelus dromedarius, during the breeding season (January - March). A portion of each raw ejaculate was evaluated for sperm physical and morphological traits, whereas the other portion was divided into three aliquots assigned for the following liquefaction treatments: control (untreated), 0.1 mg/mL papain or 5 U/mL bromelain. All samples were diluted with Tris-lactose diluent containing the anti-enzyme E-64 to neutralize both proteases before being processed for cryopreservation. Post-thaw physical and kinematic properties of spermatozoa were analyzed using a computer-assisted sperm analysis (CASA) system. The sperm surface glycocalyx pattern was evaluated with a panel of 14 fluorescent lectins. Although bromelain was more effective in elimination of semen viscosity, there was a negative correlation between bromelain supplementation and values for the variables: normal sperm, intact acrosome and intact sperm cell membrane. Bromelain supplementation, compared to papain-treated and control samples, was positively correlated with secondary sperm abnormalities, increased straight-line velocity (VSL, μm/s) and straightness (%) of spermatozoa. Results from the glycan analysis indicated that both proteases did not affect the N-linked glycan content of the entire sperm surface, whereas the treatment with proteases induced little change in N-acetylgalactosamine and fucose terminating glycans in the tail region of the sperm. Functional studies are needed to evaluate the sperm fertility rates of bromelain- and papain-treated semen for application in camel assisted reproductive technologies.

Liquid storage of dromedary camel semen in different extenders

This study was conducted to assess the effects of commercial extenders and storage temperature on dromedary camel sperm quality during liquid preservation. In Experiment 1, ejaculates (n = five males; replicated seven times) were split and diluted with synthetic (OPTIXcell, EquiPlus, INRA96, Bioxcell or AndroMed; Experiment 1a) or egg-yolk based (Biladyl, Green buffer or Triladyl; Experiment 1b) extenders and stored for 48 h at 4 °C. In Experiment 2, split ejaculates (n = five males; replicated six times) were used to directly compare Green buffer, OPTIXcell and Triladyl extenders over 48 h of storage at 4 °C. Ejaculates collected in Experiment 3 (n = five males; replicated five times) were diluted with Green buffer or Triladyl before chilled storage for 48 h at 4 or 15 °C. Sperm kinematics, viability and acrosome integrity were assessed during liquid storage. In Experiment 1a, there was the greatest total sperm motility (TM) in the OPTIXcell group following 24 and 48 h of storage, while in Experiment 1b, there was the greatest TM after 48 h of storage with Triladyl and Green buffer. In Experiment 2, there were greater TM and viable acrosome intact spermatozoa in the Triladyl and Green buffer than with OPTIXcell group. In Experiment 3, there was a greater TM in the Triladyl than Green buffer group at 24 and 48 h of storage regardless of storage temperature (which had no effect on sperm quality). In conclusion, camel sperm have greater viability when preserved in liquid form for 48 h following dilution with Triladyl and storage at either 4 or 15 °C.

Acoustic trapping of sperm cells from mock sexual assault samples

We report the successful separation of sperm cells from a relevant composition of mock sexual assault samples using a novel acoustic differential extraction (ADE) technology. A multi-layer microfluidic device fabricated in a non-photolithographic process from glass and polydimethylsiloxane (PDMS) was capable of interfacing with custom-built instrumentation to exploit a standing acoustic wave for the trapping of individual sperm cells in a sample containing an abundance of epithelial cells. Samples were generated from buccal and vaginal swabs to mimic post-coital vaginal swabs, and processed through the ADE system followed by DNA extraction of the captured cells with amplification of DNA using a custom short tandem repeat (STR) chemistry. The prototype acoustic trapping technology was fully capable of isolating intact sperm cells from mock samples with disparate masses of male and female DNA. Other biological components were evaluated for adverse effects on sperm cell trapping, including blood, yeast, and bacteria (E. coli), and these had negligible effects on observed sperm cell trapping. Finally, we demonstrate the successful capture of sperm cells from mock samples containing a 40-fold excess in female epithelial cells over sperm cells. The effectiveness of sperm cell purification was ascertained with polymerase chain reaction (PCR) amplification of STR loci from the male fraction post separation with an 18-plex amplification kit, which resulted in male-only profiles.

EFFECT OF SUPPLEMENTATION OF OMEGA-3 FATTY ACIDS ON BLOOD PARAMETERS AND SEMEN QUALITY OF FRIESIAN BULLS

The aim of this work was to evaluate the effects of flaxseed oil supplementation as a source of omega-3 fatty acids (OFA) on some blood parameters, semen quality and testosterone level in male Frisian bull. A total of 30 Friesian bulls (14.2 ± 0.16 month of age and 265±15 kg body weight) were divided into three equal groups according to age and live body weight. Bulls in the 1st group (G1) were supplemented with 2% dray matter intake (DMI) flaxseed oil as a source of OFA, while those in 2nd group were supplemented with 4% DMI flaxseed oil as a source of OTA at 14 to 21 months of age (G2). Bulls in the 3rd group were fed a basal diet and considered as a control group (G3). The obtained results revealed that OFA administrated animals had a significant (P&lt; 0.05) inducing effect on serum levels of total protein, globulin, glucose, high density lipoprotein (HDL) and triglyceride compared to the control group. No significant changes were noticed in albumin levels among the three groups. However, the levels of total cholesterol, urea and low density lipoprotein (LDL) were significantly lower in bulls received OFA than the control group. Omega-3 fatty acids treatment has a significant positive effect on the semen characteristics and lower abnormality in G2 and G3 than in G1. G2 and G3 also showed a significant higher intact spermatozoa cell membrane than in G3 by about 9.68 and 10.84%, respectively. Omega-3 treatment significantly increased blood testosterone levels to be 23,78% in G2 and 31.82% in G3 higher than that of the control. In conclusion, dietary supplementation with omega-3 in male Frisian ration improved semen quality and reproductive potentiality as well as testosterone level.Key words: Frisian bull; omega-3 fatty acids; testosterone and semen quality

Addressing the Compartmentalization of Specific Integrin Heterodimers in Mouse Sperm.

Integrins are transmembrane cell receptors involved in two crucial mechanisms for successful fertilization, namely, mammalian intracellular signaling and cell adhesion. Integrins α6β4, α3β1 and α6β1 are three major laminin receptors expressed on the surface of mammalian cells including gametes, and the presence of individual integrin subunits α3, α6, β1 and β4 has been previously detected in mammalian sperm. However, to date, proof of the existence of individual heterodimer pairs in sperm and their detailed localization is missing. The major conclusion of this study is evidence that the β4 integrin subunit is expressed in mouse sperm and that it pairs with subunit α6; additionally, there is a detailed identification of integrin heterodimer pairs across individual membranes in an intact mouse sperm head. We also demonstrate the existence of β4 integrin mRNAs in round spermatids and spermatogonia by q-RT-PCR, which was further supported by sequencing the PCR products. Using super-resolution microscopy accompanied by colocalization analysis, we located integrin subunits as follows: α6/β4-inner apical acrosomal membrane and equatorial segment; α3, α6/β1, β4-plasma membrane overlaying the apical acrosome; and α3/β1-outer acrosomal membrane. The existence of α6β4, α3β1 and α6β1 heterodimers was further confirmed by proximity ligation assay (PLA). In conclusion, we delivered detailed characterization of α3, α6, β1 and β4 integrin subunits, showing their presence in distinct compartments of the intact mouse sperm head. Moreover, we identified sperm-specific localization for heterodimers α6β4, α3β1 and α6β1, and their membrane compartmentalization and the presented data show a complexity of membranes overlaying specialized microdomain structures in the sperm head. Their different protein compositions of these individual membrane rafts may play a specialized role, based on their involvement in sperm-epithelium and sperm-egg interaction.

Comparison of Sperm Functional Parameters after Cryopreservation of Human Sperm in Three Different Media

Semen cryopreservation is an integral part of infertility treatment in the current day scenario. Recovery of an optimal number of functionally intact spermatozoa from thawed samples has always been the main objective of semen cryopreservation technology. The aim of this study was to compare of Commercial freezing media with Glycerol egg yolk citrate and Soy lecithin media for the cryopreservation of human spermatozoa. This prospective observational study was done at the department of reproductive medicine and surgery, Sri Ramachandra University during the period of August 2015 to January 2016. Male partner of the infertile couple visiting OPD for infertility treatment with normal semen quality (Normozoospermic) were included in the study. Each semen sample was divided into three aliquots and frozen with Commercial media, Glycerol Egg Yolk Citrate (GEYC) media and soy lecithin media. Semen analysis and sperm function tests before freezing and after thawing namely Acrosome reaction, Capacitation and Mitochondrial potential was compared between the groups. There was a significant decline in motility, vitality and functional parameters of the sperms compared to fresh and frozen thawed in all the groups. However, among the frozen thawed groups, the commercial media and GEYC media were comparable in motility and viability parameters;GEYC media was superior to Commercial media in functional sperm parameter maintenance.

Effect of γ-oryzanol on testicular degeneration induced by scrotal insulation in rams

The present study assessed the effects of daily supplementation with 33 mg/metabolic weight (MW) of γ-oryzanol on testicular degeneration induced by scrotal insulation in rams. Eight animals were divided into two groups: Control (subjected to scrotal insulation without treatment) and Gamma (subjected to scrotal insulation and γ-oryzanol treatment). The rams were subjected to scrotal insulation by covering the scrotum with a thermal bag for 72 h. Animals in the Gamma group received 33 mg/MW oral γ-oryzanol once-daily, beginning 7 days before insulation and continuing during insulation and for 20 days afterward, for a total treatment period of 30 days. Samples of semen and blood were collected during the experiment to perform biochemical evaluations of oxidative stress, seminal kinetics and morphology, and plasma testosterone concentrations. Ultrasound examinations of the testicular parenchyma and clinical evaluations of its consistency and the scrotal perimeter were also performed at weekly intervals. Testicular tissue was collected for biochemical analyses of oxidative stress parameters at the end of the experiment by orchiectomy. The results showed that testicular degeneration was induced by scrotal insulation, as was demonstrated by the reduced scrotal perimeter and increased in testicular flaccidity immediately after insulation. Moreover, a delayed increase in the number of hyperechoic points in the parenchyma and a delayed reduction in sperm motility were observed at 10 weeks after insulation by ultrasonography. Treatment with γ-oryzanol reduced levels of reactive oxygen species (ROS) levels in the testes, and increased the total antioxidant potential (assessed based on the ferric reducing ability (FRAP)) in week 10 and levels of lipid peroxidation (TBARS). It also increased the number of intact spermatozoa in week 3, but increased the total number of sperm defects from week 5 onwards. Although γ-oryzanol protected the semen and testes by reducing the levels of the parameters of oxidative stress evaluated herein, the other parameters studied were not improved by the treatment. In addition, supplementation with γ-oryzanol led to more morphological abnormalities in the sperm. This study presented new information on the oral administration of γ-oryzanol to rams with testicular degeneration, and described potential therapies for this pathology, which currently has no established treatment and has important impacts on reproductive health.

The role of metalloproteases in fertilisation in the ascidian Ciona robusta

In the ascidian Ciona robusta (formerly C. intestinalis type A), the mechanism underlying sperm penetration through the egg investment remains unknown. We previously reported that proteins containing both an astacin metalloprotease domain and thrombospondin type 1 repeats are abundant in the sperm surface protein-enriched fraction of C. robusta. Here we investigated the involvement of those proteins in fertilisation. We refined the sequences of astacin metalloproteases, confirmed that five of them are present in the sperm, and labelled them as tunicate astacin and thrombospondin type 1 repeat-containing (Tast) proteins. Fertilisation of C. robusta eggs was potently inhibited by a metalloprotease inhibitor GM6001. The eggs cleaved normally when they were vitelline coat-free or the inhibitor was added after insemination. Furthermore, vitelline coat proteins were degraded after incubation with intact sperm. These results suggest that sperm metalloproteases are indispensable for fertilisation, probably owing to direct or indirect mediation of vitelline-coat digestion during sperm penetration. TALEN-mediated knockout of Tast genes and the presence of GM6001 impaired larval development at the metamorphic stage, suggesting that Tast gene products play a key role in late development.

A study to determine the prefreeze motility parameters suggesting a favorable outcome

Introduction The recovery of functionally intact spermatozoa from thawed samples is dependent on the cryopreservation process and initial quality of the semen sample. Material and Methods 100 semen samples were cryofrozen by conventional slow freezing method. With reference of post thaw PR ≥0.4 (ratio of post thaw progressive motility/ pre thaw progressive motility) samples were grouped into poor freezability ejaculates (PFE, n = 19) with ratio < 0.4 and good freezability ejaculates PR ≥0.4 (GFE, n = 81). Results There was no statistical significance of survival of sperms with age of husband and sperm count prior to cryopreservation. The semen samples which had curvilinear velocity in the range of 56.47- 97.95 um/s-1, straight line velocity 42.811 - 72.649 um/s-1 and average path velocity 39.77- 70.11um/s-1 before freezing have good survival of sperms after thawing. Conclusions Assessment of the prefreeze velocity parameters by CASA to predict the survival of sperms post thawing so that proper counseling of patients regarding cryosurvival of sperms could be done during semen cryopreservation.

Influences on thermo-resistance of boar spermatozoa

The aim of this study was to investigate the influence of semen production management in 25 European boar studs on thermo-resistance (TRT) of boar spermatozoa. TRT is an important semen characteristic, easy to determine, and is used to explain variations in pig fertility. During a study period from 2013 to 2018, 905 ejaculates were analyzed for TRT on day 7 of semen storage. Statistical models showed that around 40% of the total variability in TRT could be explained by production management factors. Significant selected predictors were year, month, boar stud, boar age, dilution steps, mitochondrial activity and membrane integrity of spermatozoa, bacterial contamination, arrival temperature of ejaculates in the analysis laboratory, sperm concentration, ejaculate volume, sperm output, dose/ejaculate, and electrical conductivity of pure water for extender preparation. TRT increased during the study period. No effects were observed for breed, dilution rate, morphologically intact spermatozoa, and refractive index of extender on TRT. A holistic view of the requirements in the field of boar semen production is of great importance for future developments of individualized Hazard Analysis and Critical Control Point concepts, prognosis models for boar semen quality, and could help to improve the efficiency of AI organizations.

Effect of the Number of Mating Partners and Sperm Quality on Reproductive Success in the Domestic Cat (Felis catus)

Data on the reproductive success and reproductive behavior of the domestic cat, the most widespread representative of the family Felidae, are presented. The effects of the number of partners, mating order, and sperm quality on male and female reproductive success were assessed. The experiments showed that the fertility of female cats that mated with two partners was higher. Here, male reproductive success was less dependent on the attitude of females to the males, whereas the sperm quality (percentage of morphologically intact spermatozoa) and order of mating with the female had considerable effects on reproductive success. However, male sperm quality did not have a significant effect on the number of offspring if the mating pattern was monogamous.