Collection and freezing of equine epididymal spermatozoa

The epididymis and vas deferens store an important number of fertile spermatozoa called the extragonadal sperm reserves. These stored spermatozoa can be collected in an ultimate attempt to preserve viable spermatozoa of a critically ill or dying stallion. Epididymides are collected via routine castration. After cooled transport of the testicles and epididymides, spermatozoa are collected either by retrograde flushing or by the float-up method. Retrograde flushing usually results in a much higher sperm yield and is considered the method of choice. Epididymal spermatozoa can be frozen using standard freezing protocols

Immunogenicity of human spermatozoa

Investigation and experimental design of the study was basically aimed at developing insight into the antigenicity of spermatozoa-associated proteins. Apart from studying the natural antigenicity of washed whole spermatozoa, their immunogenicity was also demonstrated _in vitro_. The whole live spermatozoa were immobilized and agglutinated _in vitro_ by the antibodies they induced in the laboratory model - a female rabbit. A regular immunization routine induced a high titre of antisperm polyclonal antibodies. To prepare a spermatozoa specific antigen which will not produce a cross-reacting antibody against other human tissues, only the motile and live spermatozoa were selected for antigen preparation. In investigation the laboratory-bred female rabbits were used as the bioactive system of production of antisperm antibody. Agglutination of whole spermatozoa has been observed on slides. The technique though simple is highly eloquent; clumping of spermatozoa confirms the existence of antisperm antibodies in the serum under examination. The results show that nature and pattern of immobilization of active motile spermatozoa are different as observed in different graphs of immobilization. The variation in spermatozoal population in antigen distribution on individual spermatozoa is reflected in different patterns of agglutination

Automatic detection of spermatozoa for laser capture microdissection

In sexual assault crimes, differential extraction of spermatozoa from vaginal swab smears is often ineffective, especially when only a few spermatozoa are present in an overwhelming amount of epithelial cells. Laser capture microdissection (LCM) enables the precise separation of spermatozoa and epithelial cells. However, standard sperm-staining techniques are non-specific and rely on sperm morphology for identification. Moreover, manual screening of the microscope slides is time-consuming and labor-intensive. Here, we describe an automated screening method to detect spermatozoa stained with Sperm HY-LITER (TM). Different ratios of spermatozoa and epithelial cells were used to assess the automatic detection method. In addition, real postcoital samples were also screened. Detected spermatozoa were isolated using LCM and DNA analysis was performed. Robust DNA profiles without allelic dropout could be obtained from as little as 30 spermatozoa recovered from postcoital samples, showing that the staining had no significant influence on DNA recovery

Effects of the slow cooling during cryopreservation on the survival and morphology of Taiwan shoveljaw carp (Varicorhinus barbatulus) spermatozoa

Over the past decades, pollution, overfishing, and habitat degradation have driven the population size of Taiwan shoveljaw carp down markedly in Taiwan. Cryopreservation is a useful tool which could be used to maintain genetic resources to protect and preserve this endemic species. Four cryoprotectants [dimethyl sulphoxide (DMSO), dimethylacetamide (DMA), glycerol and methanol] and six freezing rates (0.5, 1, 2, 4, 8, 16 °C min-1) were tested in order to develop an optimal controlled slow-freezing protocol for Taiwan shoveljaw carp spermatozoa. Samples were subsequently examined under the scanning electron microscope to reveal whether cryopreservation had affected their ultrastructural morphology. The highest survival rate (50.1 ± 2.0%) was observed with a freezing rate of 8 °C min-1 in 1M DMSO, using SYBR-14 + PI staining. Fertility and hatching rate results using frozen-thawed spermatozoa (90.2 ± 2.2% and 22.3 ± 2.5%, respectively) were not significantly different from results with fresh spermatozoa. After cryopreservation, 21.0 ± 1.6% of frozen-thawed spermatozoa had mid-piece swelling and rupture of the head. Cryopreservation might, therefore, slightly affect Taiwan shoveljaw carp spermatozoa in terms of morphological change. However, these alterations could be compensated by using large enough numbers of normally functioning frozen-thawed spermatozoa to achieve a standard equal to fresh spermatozoa. This is the first report of successful cryopreservation of Taiwan shoveljaw carp spermatozoa using a controlled slow-cooling method

Exceptional sperm cooperation in the wood mouse

Spermatozoa from a single male will compete for fertilization of ova with spermatozoa from another male when present in the female reproductive tract at the same time. Close genetic relatedness predisposes individuals towards altruism, and as haploid germ cells of an ejaculate will have genotypic similarity of 50%, it is predicted that spermatozoa may display cooperation and altruism to gain an advantage when inter-male sperm competition is intense. We report here the probable altruistic behaviour of spermatozoa in an eutherian mammal. Spermatozoa of the common wood mouse, Apodemus sylvaticus, displayed a unique morphological transformation resulting in cooperation in distinctive aggregations or 'trains' of hundreds or thousands of cells, which significantly increased sperm progressive motility. Eventual dispersal of sperm trains was associated with most of the spermatozoa undergoing a premature acrosome reaction. Cells undergoing an acrosome reaction in aggregations remote from the egg are altruistic in that they help sperm transport to the egg but compromise their own fertilizing ability

In Vitro Fertility of Post-thawed Epididymal Ram Spermatozoa After Storage at 5 °C Before Cryopreservation

This study addressed the effects of storage duration of epididymides at 5 °C before sperm collection and their fertility after cryopreservation in vitro. Spermatozoa from one of the testes pairs were immediately collected, evaluated and frozen (control group). The remaining epididymides were cooled to 5 °C and stored for 24, 48, 72, and 96 h (experimental groups), after which spermatozoa were collected and frozen as in the control group. Before and after thawing, sperm motility, sperm viability and plasma membrane integrity were assessed. The fertilizing ability of frozen-thawed spermatozoa of each group was evaluated by in vitro fertilization of matured sheep oocytes. Sperm quality (sperm motility, viability, and plasma membrane integrity) at collection and after cryopreservation decreased as the duration of the epididymal storage interval increase (P < 0.05). The motility decreased steadily along the studied time periods. Although, the fertilizing ability of post-thawed epididymal spermatozoa gradually decreased as the storage period was prolonged, the spermatozoa collected from the cauda epididymides stored at 5 °C for up to 96 h were able to fertilize 16%-65% of oocytes in vitro. Results of the present study showed that ram epididymal spermatozoa survive in storage at 5 °C for up to 96 h. These spermatozoa maintain their fertilizing ability and may be suitable for use in IVF and other assisted reproductive procedures

Chemotaxis of Arbacia punctulata spermatozoa to resact, a peptide from the egg jelly layer

Resact, a peptide of known sequence isolated from the jelly layer of Arbacia punctulata eggs, is a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for millimolar external calcium. A. punctulata spermatozoa do not respond to speract, a peptide isolated from the jelly layer of Strongylocentrotus purpuratus eggs. This is the first report of animal sperm chemotaxis in response to a defined egg-derived molecule

Studies on the sperm reservoir of the pig oviduct

During sperm transport in the female pig a proportion of spermatozoa are arrested, often for 24 h or more, in a particular segment of the utero-tubal junction (UTJ) and the adjacent tubal isthmus, where a sperm reservoir (SR) is built up. The SR maintains, via its lining epithelium and the fluid it produces, sperm viability pre-ovulation. It also controls the release of potentially fertilising spermatozoa so that only a small sub-population reaches the site of fertilisation, thus diminishing the risk of polyspermy. In vitro research has focused on sperm binding as the main mechanism of sperm storage, sperm release and modulation of capacitation, but little attention has hitherto been paid to the isthmic oviductal fluid (IOF), its composition and the control of capacitation in vivo. This thesis aimed to study the intra-luminal milieu of the SR in sexually cycling gilts and sows, its contents of glycosaminoglycans (GAGs) — particularly of the non-sulphated hyaluronan (HA) — and the presence and expression of the specific HA receptor CD44 and of HA synthases. Both non-inseminated (controls) and inseminated animals were studied during specific moments of oestrus, in relation to spontaneous ovulation. Ultimately, the study aimed to determine the capacitation status of SR-stored spermatozoa and the effect of IOF and HA on capacitation of the SR-stored spermatozoa. The results showed that SR-stored spermatozoa are entrapped in a mucus-like IOF pre-ovulation. This IOF contains fluctuating levels of sulphated GAGs and HA. Hyaluronan is synthesised in the lining epithelium by HA synthase 3 (has3). Both the ligand and the specific HA-receptor CD44 were particularly present in the deep furrows of the SR, where most spermatozoa are trapped. Massive sperm capacitation does not occur in vivo in the porcine SR under spontaneous standing oestrus, particularly during pre- and peri-ovulation, but SR spermatozoa capacitate if exposed to the effector bicarbonate. Exposure of SR spermatozoa to IOF (or its component HA) in vitro was seen to reverse the bicarbonate influence during pre- and peri-ovulation but to potentiate capacitation post-ovulation, suggesting an active role for the intra-tubal fluid and/or HA in modulating sperm capacitation in pigs. The findings support the concept that the oviductal SR keeps the potentially fertile spermatozoa viable and uncapacitated during their pre-ovulatory arrest. The findings may help improve sperm preparation protocols for porcine in vitro fertilisation (IVF) and preservation of boar semen

Corner accumulation behavior of spermatozoa in microchannels

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.In this study, microfluidic approaches and fluorescence microscopy were used to study cross-sectional distribution of bull spermatozoa in a rectangular microchannel. The results indicate a strong corner accumulation behavior of bull spermatozoa in a rectangular microchannel. Results indicate that 74% of spermatozoa accumulate near boundaries and only 26% of spermatozoa are bulk swimmers. Furthermore, 66% of wall swimmers are corner swimmers. The distinction and quantification of wall vs. corner vs. bulk swimmers was enabled by the unique head-on microchannel imaging approach applied here

Maintenance of Constant Wave Parameters by Sperm Flagella at Reduced Frequencies of Beat

1. Treatment of Ciona spermatozoa with low concentrations of Triton X-100 (less than 0·01 %) causes them to beat at lower than normal frequencies. The wavelength of the flagellar bending waves remains constant over the range from 10 to 40 Hz. There is a small increase in wavelength at lower frequencies; in the range of 1·5-6·2 Hz, the wavelength averaged 114% of the normal value for Ciona spermatozoa. The angle of bend of the bent regions of the flagellar bending waves remained constant within ± 10% over this range of frequencies. 2. Decapitated sperm flagella from Lytechinus beat at a continually declining frequency as they exhaust their content of ATP. Both wavelength and bend angle retain normal values until the frequency falls below about 8 Hz. Both parameters increase at lower frequencies, with a sharp increase below 3 Hz. 3. ATP-reactivated spermatozoa from Lytechinus show relatively small changes in wavelength and bend angle as the frequency is varied over the range from 5 to 25 Hz by varying the ATP concentration. 4. Constancy of wavelength over a wide range of frequencies is consistent with the hypothesis that wavelength is determined by the relative values of viscous bending resistance within a flagellum and external viscosity. 5. No satisfactory explanation is available at present for the constancy of bend angle over a wide range of frequencies nor for the changes in wave parameters which are observed at low frequencies