Semen Fractions Research Articles

Limited shedding of an S-InDel strain of porcine epidemic diarrhea virus (PEDV) in semen and questions regarding the infectivity of the detected virus

PEDV is mainly transmitted by the oro-fecal route although PEDV shedding in semen has already been shown for an S-non-InDel PEDV strain infection. The aim of this study was to determine if PEDV can be shed in semen from SPF (specific pathogens free) boars infected by a French S-InDel PEDV strain (PEDV/FR/001/2014) and in case of positive semen to determine the infectivity of that semen. Both infected boars had diarrhea after inoculation and shed virus in feces. PEDV genome was also detected by RT-qPCR in the sperm-rich fraction of semen (6.94 × 103 and 4.73 × 103 genomic copies/mL) from the two boars infected with the S-InDel PEDV strain but only once at 7DPI. In addition, PEDV RNA in Peyer’s patches and in mesenteric lymph nodes was also present for the two inoculated boars. The PEDV positive semen (S-non-InDel and S-InDel) sampled during a previous trial and in this boar trial were inoculated to six SPF weaned pigs. The inoculated piglets did not seroconvert and did not shed virus throughout the duration of the study except for one pig at 18 DPI. But, PEDV could be detected in intestinal tissues such as duodenum, jejunum and jejunum Peyer’s patches by RT-qPCR except for one pig. Even if PEDV genome has been detected in semen, experimental infection of piglets with positive semen failed to conclude to the infectivity of the detected PEDV.

Potent and rapid activation of tropomyosin-receptor kinase A in endometrial stromal fibroblasts by seminal plasma.

Seminal plasma (SP), the liquid fraction of semen, is not mandatory for conception, but clinical studies suggest that SP improves implantation rates. Prior in vitro studies examining the effects of SP on the endometrium, the site of implantation, surprisingly revealed that SP induces transcriptional profiles associated with neurogenesis. We investigated the presence and activity of neurogenesis pathways in the endometrium, focusing on TrkA, one of the canonical receptors associated with neurotrophic signaling. We demonstrate that TrkA is expressed in the endometrium. To determine if SP activates TrkA signaling, we isolated the two most abundant endometrial cell types-endometrial epithelial cells (eEC) and endometrial stromal fibroblasts (eSF)-and examined TrkA activity in these cells after SP exposure. While SP only moderately activated TrkA in eEC, it potently and rapidly activated TrkA in eSF. This activation occurred in both non-decidualized and decidualized eSF. Blocking this pathway resulted in dysregulation of SP-induced cytokine production by eSF. Surprisingly, while the canonical TrkA agonist nerve growth factor was detected in SP, TrkA activation was principally induced by a 30-100-kDa protein whose identity remains to be established. Our results show that TrkA signaling is highly active in eSF and is rapidly induced by SP.

Evidence of porcine epidemic diarrhea virus (PEDV) shedding in semen from infected specific pathogen-free boars

In 2013, PED emerged for the first time in the United States (US). The porcine epidemic diarrhea virus (PEDV) spread quickly throughout North America. Infection with PEDV causes watery diarrhea and up to 100% mortality in piglets, particularly for highly pathogenic non-InDel strains circulating in the US. PEDV is mainly transmitted by the fecal–oral route. Transmission via the venereal route has been suspected but not previously investigated. The aim of the study was to determine if PEDV could be detected in semen from infected specific pathogen-free (SPF) boars inoculated with a PEDV US non-InDel strain suggesting venereal transmission may occur. Two boars orally inoculated with PEDV showed clinical signs and virus shedding in feces. Transient presence of the PEDV genome was detected by RT-qPCR in the seminal (5.06 × 102 to 2.44 × 103 genomic copies/mL) and sperm-rich fraction of semen (5.64 × 102 to 3.40 × 104 genomic copies/mL) and a longer duration of viral shedding was observed in the sperm-rich fraction. The evidence of PEDV shedding in semen raises new questions in term of disease spread within the pig population with the use of potentially contaminated semen.

Effect of oviduct and follicular fluids on ram sperm capacitation and acrosome reaction in vitro

The present study was designed to study the influence of different concentrations of oviduct fluid (OF) and follicular fluid (FF) on ram sperm capacitation and acrosome reaction in vitro. Forty semen samples were collected from three Barki rams throughout the period of study (10 weeks). Fresh semen was evaluated, layered under S-TALP (sperm Tyrode’s albumin lactate pyruvate) medium and subjected to swim up techniques. Split fractions of semen were incubated in media enriched with different levels of OF (10, 20, 40, 50 and 75 µL/mL) or FF (10, 20, 40, 50, 100, 150 and 200 µL/mL). Best concentrations were compared and used to evaluate the ram sperm functions including progressive motility, hyperactivity and acrosome reaction. The present findings showed a significant increase in individual motility percentage (IM %) when ram spermatozoa were treated with 10 and 50 µL of oviduct fluids as compared to the other treatments after 1 h of incubation. Addition of 50 µL/mL of OF or FF had beneficial effect on sperm hyperactivity after 2 and 1 h incubation respectively. Furthermore, addition of 50 µL/mL oviduct fluid to ram sperm maintained significantly (P < .05) higher total acrosome reaction (AR %) after 3 h of incubations than those observed in other groups. In conclusion, treatment of ram spermatozoa with 50 µL/mL of OF or 40 µL/mL of FF for 3–4 h incubation respectively was considered the best level of oviduct or follicular fluid to be used for IVF.

An endeavor to improve longevity of cryopreserved equine sperm

Exposure of sperm cells to the oxidative stress pending hypothermic storage of semen has been suggested to be responsible, in part, for the decline of their motility and fertility. This study was conducted to evaluate the in-vitro effects of antioxidants (AOs) and / or caffeine on longevity of cryopreserved stallion spermatozoa. Aliquots from the gel-free fraction of semen ejaculates (n=12), collected from 5 Arabian stallions (9-18 years old) of unknown sperm freezability, were mixed 1:1 with a Tris-egg yolk extender (TEYE), centrifuged at 500 χ g for 5 min and sperm cells were frozen in the form of 0.25-ml concentrated pellets after 2-step addition of TEYE supplemented with or without AOs (0.50 mg /ml Na pyruvate, 1 mg / ml Na thiosulfate, 5 mg / ml bovine serum albumin, 0.15 mg / ml zinc chloride and 0.50 mg / ml ferulic acid). The final pre-freeze concentrations of glycerol and sperm cells were 5% and 562-924 χ IO6 / ml, respectively. Frozen pellets from non-AOs and AOs-treated sperm were thawed in a Tris-citric acidglucose solution (40°C) containing 0, 0.49, 0.97 or 1.94 mg / ml caffeine and incubated (140-230 χ IO6 sperm / ml) at 30°C for 3 h. Sperm progressive motility (%) was assessed after centrifugation, before freezing and after 0, 1, 2 and 3 h of thawing. The results revealed significant (P&lt;0.05) effects of sperm treatments only on post-thaw motility. Neither AOs alone nor caffeine alone could significantly ameliorate the maintenance of sperm motility. AOs plus 0.97 or 1.94 mg / ml caffeine were the superior supplements in improving the longevity of stallion spermatozoa.

Induction of immune-related gene expression by seminal exosomes in the porcine endometrium

Seminal plasma (SP) is considered as a vehicle to carry sperm into female reproductive tract, of which functions have not been completely understood. This study aimed to identify the function of seminal exosomes on porcine endometrium. Exosomes were isolated from the sperm-rich fraction of boar semen and were confirmed by the expression of exosome marker HSP70 and size distribution using nano-sight tracking analysis. Porcine endometrial epithelial cells (EECs) were then treated with seminal exosomes, and RNA extracted were subjected to global expression analysis. Transcripts related to “immune response”, “inflammatory response” and their associated signaling pathways were up-regulated in EECs treated with seminal exosome, whereas those associated with “steroid biosynthesis”, “metabolic pathways” and “T cell differentiation” were down-regulated. The decrease in PMVK, SC5D, INSIG1, HSD17B7, NSDHL, HMGCR, SQLE and FDFT1, and increase in CCL20, TNFSF15, AMCFII, CXCL2 and CXCL8 were also found in the endometrium from the naturally mated pigs. Moreover, changes in exosome-induced CYP24A1, EBP, CCL20, AMCFII and IL1A expression were not regulated by the exosome removed SP. These observations indicated that exosomes present in SP are involved in the immune-related gene regulation in the uterus, which could pave the passage for sperm and possibly fertilized eggs.

Cellular Targets and Receptor of Sexual Transmission of Zika Virus.

What is the mechanism of sexual transmission of Zika virus (ZIKV)? By utilizing exquisite reverse transcriptase-initiated in situ polymerase chain reaction (RT-in situ PCR), which enables an improved visualization of spermatozoa's subcellular compartment, we precisely localized the mid-piece of sperm that carry receptors for ZIKV. ZIKV is transmitted sexually and recent studies have verified ZIKV presence in semen of previously Zika-infected patients for >6-month postinfection when ZIKV had disappeared from blood, saliva, and urine. Strong serial analyses of various body fluids suggest that ZIKV can be transmitted between sexual partners. Currently, there is limited information on the association of the virus with human semen cell types that may carry the virus. Analyses were carried out to localize ZIKV for subcellular localization of ZIKV on cell types. The Tyro3 receptor for ZIKV was colocalized by dual immunocytochemistry with specific monoclonal antibodies. Three semen specimens were purchased from a commercial sperm bank. Motile sperm was separated from nonmotile cells by the "swim-up" technique. Each of the semen fractions was infected with ZIKV at the multiplicity of infection of 0.1.0 and 1.0 and evaluated for the primary targets of ZIKV in the semen cells by RT-in situ PCR and confirmed by real-time RT-PCR. ZIKV was present primarily at the mid-piece of mature spermatozoa in about 30% of the sperm. In addition, we determined that Tyro3 receptors, primarily expressed on mid-piece of human spermatozoa, play a role in ZIKV-binding and entry into spermatozoa. Our data strongly suggest a potential sexual/horizontal route of transmission for ZIKV primarily via infected sperms; most likely ZIKV enters the sperm via the Tyro3 receptor found at the mid-piece of the mature spermatozoa. We are uncertain as to what phase of spermatogenesis, that in human takes about 120 days, sperms are permissive to ZIKV. If permissiveness was very early during spermatogenesis males could be infectious for ∼120 days after the disappearance of viremia in an infected man. Our findings bring a new focus on the current affords to develop ZIKV vaccine. Why in the presence of anti-ZIKV antibodies infected men are still able to transmit the virus sexually? We suggest that only certain subclass of immunoglobulin (Ig)G (ie, IgG4) can cross the blood-Sertoli barrier therefore, a successful vaccine must provoke a subclass of IgG can quell ZIKV inside the seminiferous tubules.

Genomic fragmentation and extrachromosomal telomeric repeats impact assessment of telomere length in human spermatozoa: quantitative experiments and systematic review.

Can differences in DNA isolation alter assessment of sperm telomere length (spTL) and do they account for conflicting results in the literature on spTL and male fertility? DNA isolation methods preferentially include or exclude short, extrachromosomal (EC) telomere-specific sequences that alter spTL measurements, and are responsible for a proportion of the disparity observed between investigations. The relationship between spTL and male fertility has become an active area of research. The results across investigations, however, have been discordant, generating a need to critically evaluate the existing body of knowledge to guide future investigations. Quantitative experiments determined the effect of DNA isolation on the integrity of sperm DNA and measures of spTL, while a systematic analysis of the current literature evaluated the effect of DNA isolation and study design on experimental outcomes. Two DNA isolation methods were compared: Genomic Tips which isolate 'High Molecular Weight' (HMW) DNA exclusively, and QIAamp® DNA Mini which isolates 'Total' genomic DNA irrespective of size. DNA quality was assessed via field inversion gel electrophoresis (FIGE) and spTL was measured via terminal restriction fragment analysis. In addition, major databases in medicine, health and the life sciences were subject to a targeted search, and results were independently screened according to defined exclusion/inclusion criterion. Findings from primary articles were analyzed for concordance and study designs were compared across six moderator variables (sample size, participant age, fertility status, semen fraction, telomere population and type of analysis). HMW DNA spTL was significantly longer than spTL measured from total DNA (P < 0.01), indicating that Total DNA contained short, EC telomeric repeats that shifted downstream assessment towards shorter spTL. HMW DNA spTL reflected the length of intact, chromosomal telomeres. Major findings on spTL showed the greatest concordance amongst studies that implemented HMW DNA isolation prior to spTL assessment. Studies that utilized Total DNA varied in concordance, but outcomes were similar if (i) a comparative analysis was applied or (ii) a sample size threshold of 81 was achieved for correlative analysis. Chromosomal and EC telomeric DNA were distinguished based on outcomes of HMW DNA isolation and size. Further experiments are required to determine the nature and function of these two types of telomeric sequences. This study reveals a dramatic impact of upstream DNA processing and study design on measurements of spTL, which accounts for conflicting results in the literature. Future assessments of spTL should incorporate independent detection of chromosomal and EC telomeric DNA and specific experimental planning. This study was funded by CReATe Fertility Centre, Toronto, Ontario, Canada. The authors have declared no conflict of interest. N/A.

Sperm quality in the retrograde fraction of semen collected by rectal probe electroejaculation in the olive baboon

Sperm quality in the retrograde fraction of semen collected by rectal probe electroejaculation in the olive baboon

Reproductive biology of Pêga Jackasses: characteristics of sperm-rich fraction collected using the split-ejaculate method

ABSTRACT The aim of this study was to evaluate the physical and morphological characteristics of the sperm-rich fraction of jackass semen. To this end, 130 ejaculates from five Pêga jackasses were collected using an open model artificial vagina. The sperm-rich fraction was collected using the split-ejaculate method and assessed for the number of mounts/ejaculate, for physical and morphological characteristics of the semen and number of doses produced/ejaculate. It was observed that all characteristics evaluated differed among the five jackasses, except for the head defect rates. The mean values obtained for the jackass sperm-rich fraction collected were: number of mounts/ejaculate - 1.27; semen volume - 20.21mL; motility - 84.53%; vigor - 4.46; motility after dilution - 80.10%; sperm concentration/mL - 894.38 x 106; total sperm/ejaculate - 16.14 x 109; number of insemination doses/ejaculate (400x106 motile sptz) - 33.39; number of insemination doses/ejaculate (800 x 106 motile sptz) - 16.69; and percentage of normal sperm - 90.46%. Thus, in the present experiment the split-ejaculate method using an open artificial vagina worked well with the jackasses, and the sperm-rich fraction of the ejaculate of Pêga jackasses had high quality and sperm concentration, allowing its use for semen processing without reducing the number of insemination doses produced per ejaculate.

Lessons learned in andrology: looking at the spermatozoa in context.

Lessons learned in andrology: looking at the spermatozoa in context.

In vitro Evaluation of Short-term Preserved Stallion Semen in Different Doses and Temperatures

This study was conducted to evaluate the effects of different temperatures and doses on stallion sperm survival after 24 h and 48 h short-term storage. Ejaculates from four warm-blooded stallions were used in this study. Ejaculates were collected by an open-ended artificial vagina which separates fractions of semen into sterile plastic cups. Only the second and third fractions (spermatozoa-rich fractions) were used to obtain spermatozoa for the experiment. Semen was diluted with INRA 96 extender to final concentrations of 50, 100, 200 and 400x106sp/ml. Diluted semen were stored at different temperatures (2, 4 or 8°C) for 24-48 h. After 24 h and 48 h cooled storage, spermatozoon motility, abnormal spermatozoon ratio and membrane integrity were evaluated. According to results the highest motility values (52.5% and 55%) were determined at 50 and 100 x106sp/ml concentration at 4°C after 24 h cooled storage. After 48 h of storage, the highest motility was obtained from semen stored at 8°C. Abnormal spermatozoon and membrane integrity values were not significantly different after cooled storage. Consequently, it was determined that semen with 50 and 100 million spermatozoa/ml concentration stored at 4°C for 24 h and semen with 50, 100, 200, 400 million spermatozoa/ml concentration stored at 8°C for 48 h gave better results with respect to motility than the others.

Morphological evaluation of sperm from infertile men selected by magnetic activated cell sorting (MACS)

Electron microscopy analysis performed in five infertile human subjects after sperm selection by swim-up followed by magnetic activated cell sorting (MACS) demonstrated a decrease in the number of spermatozoa with characteristics compatible with cell death. However, no significant differences were found when the swim-up/MACS semen fraction was compared with swim-up fraction alone.

Success in pregnancy through intrauterine insemination at first cycle in 300 infertile couples: an analysis.

The aim of this article was to determine digital levels of the association of factors of pregnancy success after the first cycle of intrauterine insemination (IUI) with 300 infertile couples. The IUI procedure was followed at 36h after triggering the ovulation, if at least one follicle measured >15mm. Endometrium thickness (ET) and serum luteinizing hormone (LH) levels were measured at day 10 for each patient. The post-wash total motile fraction (TMF) of semen of the partner of each patient was also evaluated. The principal component analysis (PCA) was done with the data to quantify the associations of related factors. The clinical pregnancy rate of first cycle IUI attempts was 17.3%, observed in females, aged 20-39years and men with TMF >5 million spermatozoa. The ovarian stimulation enabled the development of follicles measuring >16mm, with LH levels <10mIU/L and ET >5mm for success. The PCA revealed that with the female-age parameter, three factors, NF, ET, and LH were related in the component 1; similarly, NF, LH, and RFS were related in component 2; age, NF, ET, LH, LFS, and TMF were related in component 3; and NF, ET, LH, RFS, and LFS in component 4 were related, i.e., the best correlation. Associated principal determinative factors, LH, female-age, NF and LFS values were highly significant, but the factors, ET, RFS and TMF were statistically insignificant for success through IUI in pregnancy.

Sperm Hy-Liter™: An effective tool for the detection of spermatozoa in sexual assault exhibits

A fluorescence-based assay specifically targeting human spermatozoa was tested and optimized for best staining results using a variety of mock sexual assault samples. Swab clippings versus whole swabs were evaluated for best sample preparation and to simplify workflow (direct application versus swab extraction). The practicality and sensitivity of Sperm Hy-Liter™ was compared to our current phase contrast microscopy protocol for searching for the presence of spermatozoa. Sperm Hy-Liter™ was more sensitive than phase contrast microscopy and was able to detect spermatozoa more effectively in actual sexual assault samples (recent [N=240] or 24 years old [N=4]) containing few spermatozoa. Correlations were drawn between the Sperm Hy-Liter™ spermatozoa counts and the AmpFlSTR(®) Profiler(®) Plus male profiles generated from the sperm cell DNA fractions of semen containing swabs and swab clippings. In addition, recovered spermatozoa from Sperm Hy-Liter™-stained slides with greater than 40 spermatozoa produced full STR male profiles in 20.3% of slides tested and partial STR male profiles in 52.8% of slides tested. The adoption of Sperm Hy-Liter™ offers a means to standardize and improve the efficiency of the microscopic screening of sexual assault evidence.

A Comparative Study of Distribution of Protein and Cholesterol in Various Fractions of Human Semen from Infertile and Fertile Subjects

ABSTRACTAimTo elucidate the concentration of the protein and cholesterol in different fractions of human semen from different infertile categories and comparing them with the fertile group.Materials and methodsThe human semen was collected from different infertile categories including oligoasthenospermia, asthenospermia, azoospermia, normospermia, oligospermia and fertile group. Immediately after collection, the semen analysis was done as per WHO standard protocols. After that, the semen was centrifuged to get the different fractions. Four main fractions were obtained, (1) spermatozoa, (2) debris or material that precipitates at 12 K rpm for 10 minutes, (3) prostasomes which was precipitated at 20K rpm for 120 minutes, (4) seminal plasma. The protein concentration was done by Lowry's method and cholesterol was estimated by diagnostic kit.ResultsSodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS PAGE) was run for all the categories of semen samples for their seminal plasma and the fertility associated protein was identified. A significant difference was found in the concentration of proteins in all subfractions when compared between control and infertile categories. Almost 86% of the protein was recovered from soluble fraction. In case of azoospermia, the protein content was very low when compared with fertile group. Seminal plasma proteins were visualized by silver staining. The molecular weight of the protein bands were ranging from 6.5 to 205 kDa. The band with molecular weight around 55 kDa was found to be missing in case of oligoasthenospermia. This particular protein is said to be fertility associated protein. The content of cholesterol for different subfraction of the human semen samples from infertile and fertile samples was compared. A wide range of cholesterol was recovered from prostasomes, that too purified.ConclusionA thrive study have to be done in all the subfractions of the semen irrespective of the category of samples to know the exact function of the each subfractions in terms of protein and cholesterol distribution.How to cite this articleSundaram V, Srinivas MS, Rao KA, Pathy MR, Sridharan TB. A Comparative Study of Distribution of Protein and Cholesterol in Various Fractions of Human Semen from Infertile and Fertile Subjects. Int J Infertility Fetal Med 2012;3(3):78-82.

Distribution of α‐ and δ‐Tocopherols in Seminal Plasma and Sperm Fractions of Men With Normal and Abnormal Semen Parameters

The aim of this study was to investigate the presence of the different isoforms of tocopherol (T) in seminal plasma (P) and in the sperm fractions of individuals with abnormal (group 1) and normal (group 2) sperm parameters; the relationships between these isoforms and conventional sperm parameters were also explored. Two vitamin E homologues, α-T and δ-T, were identified in the semen of all participants. Although α-T and δ-T concentrations were similar in the semen of the 2 groups, group 1 showed a lower α-T ratio (S/P) (0.90 vs. 1.20, P < .001) and δ-T ratio (0.86 vs 1.13, P = .007) than group 2. In addition, both T ratios were correlated with the percentage of viable cells, detected by eosin staining. These results suggested that α-T and δ-T are not homogeneously distributed in the semen fractions; in normal semen they are more concentrated in the sperm membrane, whereas in abnormal semen the damaged sperm cells may release both Ts in the plasma. To verify whether sperm membrane breakage could alter α-T and δ-T distribution between the seminal plasma and the spermatozoa, normal sperm samples were sonicated; after sonication a consistent sperm plasma membrane fragmentation, highlighted by transmission electron microscopy, and a concomitant release of α-T and δ-T were observed. In conclusion, the Ts coupled directly with the sperm membrane seem to play the main protective role in the semen, and the release of α-T and δ-T in the P fraction is probably an index of lower antioxidant power and sperm quality.

DNA methylation-based forensic tissue identification

Identifying the source tissue of biological material found at crime scenes can be very informative in a number of cases. Despite their usefulness, current visual, catalytic, enzymatic, and immunologic tests for presumptive and confirmatory tissue identification are applicable only to a subset of samples, might suffer limitations such as low specificity, lack of sensitivity, and are substantially impacted by environmental insults. Moreover these assays are incompatible and thus cannot be multiplexed. Thus they are less amenable to automation. In addition their results are operator-dependent. A better alternative approach is tissue identification based on messenger RNA (mRNA) or microRNA (miRNA); however, RNA is not as stable as DNA, and requires the use of non-standard procedures by forensic laboratories. Herein a DNA-based assay is described that enables tissue identification based on detection of tissue-specific methylation patterns. DNA samples are subjected to digestion by a methylation-sensitive restriction endonuclease followed by multiplex amplification of specific genomic targets with fluorescent-labeled primers, capillary electrophoresis of amplification products, and automatic signal analysis by dedicated software, yielding the source tissue of the sample. The single tube assay was designed for easy integration by forensic laboratories (as the assay utilizes the same platforms as current forensic STR profiling). The system is fully automatable, provides operator-independent results, and allows combining tissue identification with profiling in a single procedure. The assay was tested on 50 DNA samples from blood, saliva, semen, and skin epidermis, and the source tissue was successfully identified in all cases. Detection of semen and DNA profiling were combined into one assay and the ability to detect mixtures of semen and saliva in various ratios was demonstrated. The assay correctly detected semen in all samples where it was present, and the calculated percentage of semen was comparable to the fraction of semen in the samples. The results demonstrate that methylation-based tissue identification is more than a proof-of-concept. The methodology holds promise as another viable forensic DNA analysis tool for characterization of biological materials.

Hepatitis C virus RNA detection in different semen fractions of HCV/HIV-1 co-infected men by nested PCR

Objective The aim was to evaluate, by nested PCR, the prevalence of hepatitis C virus (HCV) RNA in seminal plasma in different semen fractions of HCV/HIV-1 co-infected men. Study design This study enrolled 16 HCV/HIV-1 infected men. A total of 16 seminal samples and 16 blood samples were tested for the presence of HCV-RNA. HCV-RNA in blood plasma was quantified by Amplicor HCV Monitor Test version 2.0 and HCV-RNA detection in seminal plasma, non-spermatozoa cells (NSCs), spermatozoa pellet and swim-up was investigated by nested PCR. Results Thirteen blood plasma samples were positive for HCV-RNA. HCV-RNA was detectable in seminal plasma and in non-sperm cells, but not detectable in spermatozoa samples, neither before nor after swim-up. One of the two patients whose seminal plasma tested positive at nested PCR had undetectable HCV virus in blood plasma. Conclusions HCV-RNA can be found in seminal plasma and non-sperm cells but not in spermatozoa before and after swim-up. We observed HCV-RNA in the semen of an aviremic man. According to these findings we suggest that sperm washing should be performed for each semen sample of HCV patients before assisted reproduction techniques.

Semen cryopreservation in the Indian rhinoceros (Rhinoceros unicornis)

The objective was to identify an extender and cryoprotectant combination for Indian rhinoceros ( Rhinoceros unicornis) sperm that yielded high post-thaw sperm quality. Male Indian rhinoceroses ( n = 6; 7.5–34 yr old) were anesthetized and subjected to a regimented electroejaculation procedure (75–100 mAmps; 4–10 volts; 7–150 stimuli; total of 10 electroejaculation procedures). High quality semen fractions from each ejaculate were divided into four aliquots and a 2 x 2 factorial design used to compare the effect of two sperm extenders (standard equine [EQ] and skim milk-egg-yolk-sugar [SMEY]), and two cryoprotectants (glycerol and dimethylsulfoxide [DMSO]). Cyropreserved samples were thawed and assessed for motility, viability and acrosome integrity over time. Electroejaculate fractions processed for cryopreservation had high sperm concentration (516 × 10 6/mL) and motility (79%). Post-thaw sperm characteristics were higher ( P < 0.05) when semen was cryopreserved in EQ versus SMEY. Post-thaw motility of sperm cyropreserved in EQ averaged 50–55% compared to 22–37% in SMEY, with no significant differences in sperm characteristics of samples cyropreserved in glycerol and DMSO. In conclusion, sperm collected from Indian rhinoceroses via electroejaculation were cryopreserved using EQ extender with either glycerol or DMSO; post-thaw quality was adequate for use in assisted reproductive procedures.