Raw Semen Samples Research Articles

Dead spermatozoa in raw semen samples impair in vitro fertilization outcomes of frozen-thawed spermatozoa

To evaluate the influence of dead spermatozoa present in raw semen samples before and during freezing on the functionality and fertilization ability of frozen-thawed spermatozoa. Cryopreservation of raw semen samples with different known proportions of dead spermatozoa: native semen samples (<10%) and samples with 25%, 50%, and 75% dead spermatozoa. A university-based veterinary andrology laboratory. Five healthy and sexually mature boars. Sperm killed by three fast-freezing cycles. Assessment of intracellular generation of reactive oxygen species (ROS), nuclear DNA fragmentation, in vitro fertilization (IVF), and embryo development. High proportions of dead spermatozoa in raw semen samples before and during freezing induce statistically significantly increased ROS generation and nuclear DNA fragmentation in frozen-thawed spermatozoa. These dysfunctional changes resulted in low ratios of in vitro penetrated oocytes and healthy developing embryos. A high proportion of dead spermatozoa present in raw semen samples before and during freezing negatively influences the functionality and IVF outcomes of frozen-thawed spermatozoa.

Effect of centrifugation and sugar supplementation on the semen cryopreservation of captive collared peccaries ( Tayassu tajacu)

The present study is aimed at evaluating the effect of centrifugation for seminal plasma removal and the supplementation of fructose or glucose to the Tris-based extender on the kinematic patterns of the motility parameters of frozen–thawed semen obtained from captive collared peccaries ( Tayassu tajacu). Semen samples ( n = 14) were collected from 10 sexually mature male collared peccaries by electroejaculation. These samples were further evaluated for parameters such as motility, vigor, sperm viability, membrane integrity, and sperm morphology. The samples were divided into four aliquots, and only two of these aliquots were centrifuged. The semen aliquots (centrifuged and raw semen samples) were diluted in Tris-based extenders supplemented with fructose or glucose. Egg yolk (20%) and glycerol (3%) were added to all the samples which were cryopreserved in liquid nitrogen and thawed at 37 °C/1 min. The frozen–thawed semen was evaluated for the same parameters described for the fresh semen. On the other hand, the kinematic motility patterns were evaluated by a computer-aided system. After thawing, it was observed that the values for the total sperm motility were around 30% for all the samples. A negative effect of centrifugation was verified for parameters such as sperm morphology, linearity, straightness, and beat cross frequency ( P < 0.05). However, no differences between fructose and glucose were verified for any semen end point ( P > 0.05). In conclusion, it is not recommended to centrifuge the ejaculates from collared peccaries prior to conducting the cryopreservative procedures using a Tris-based extender supplemented with fructose or glucose.

The effect of semen processing on sperm DNA integrity: comparison of two techniques using the novel Toluidine Blue Assay

Objective The first goal of this study was to determine the effect that semen processing has on sperm DNA integrity. The second goal was to assess which processing technique (modified swim-up versus density gradient centrifugation) results in a superior sample. DNA integrity was measured using a novel Toluidine Blue Assay. Study design Side-by-side comparison. Materials and methods Raw semen samples were collected from thirty-two male individuals and scored for routine semen analysis. Prior to discarding the specimens identical aliquots were divided and processed by density gradient centrifugation and a modified swim-up technique. The Toluidine Blue Assay was used to analyze raw and processed samples. Results Both density gradient centrifugation and the modified swim-up improved DNA quality compared to the unprocessed sample. However, the modified swim-up technique proved superior. Conclusions The swim-up technique generates a sperm sample with better DNA integrity. Should DNA integrity correlate with better pregnancy rates in IUI and IVF, respectively, the swim-up may be the sperm processing technique of choice for these procedures.

Processing of semen can result in increased sperm DNA fragmentation

Processing of semen for assisted reproductive technologies (ART) entails a number of procedures that include semen liquefaction, removal of seminal plasma by centrifugation, incubation, and cryopreservation. The results of this study indicate that incubation of semen at room temperature and semen cryopreservation can result in increased levels of sperm DNA fragmentation.

Molecular detection of BHV-1 in artificially inoculated semen and in the semen of a latently infected bull treated with dexamethasone

Two polymerase chain reaction (PCR) assays specific for glycoprotein B ( gB) and glycoprotein E ( gE) gene detection, respectively, were adopted for the detection of bovine herpesvirus-1 (BHV-1) in naturally infected bulls. The methods were tested on bovine semen artificially inoculated with BHV-1 and were compared with an optimised virus isolation method. Raw and extended semen samples were diluted in minimal essential medium (MEM) and spiked with equal dose of BHV-1. The extended semen was found to be more toxic for the cells than the raw semen, while the viral DNA could be detected by the PCR method in all tested dilutions of raw and extended semen samples. The sensitivity of both methods was compared also for BHV-1 detection in semen, nasal swabs and leucocytes of a seropositive bull in a different time period after virus reactivation with dexamethasone treatment. The sensitivity of virus detection by the PCR method was equivalent to that of virus isolation in cell culture. However, PCR was shown to be faster and easier to perform and may be a good alternative to virus isolation especially when bovine semen has to be screened for BHV-1 prior to artificial insemination.

Sperm survival and heterogeneity are correlated with fertility after intrauterine insemination in superovulated ewes

Efficient animal production involves accurate estimations of fertilizing ability. One key factor is the plasma membrane of the sperm cell, which is actively involved in the cascade of events before oocyte fusion. Many methods are used to analyze the characteristics of this membrane, including partition in aqueous two-phase systems which is an efficient method to analyze sperm surface changes accounting for loss of viability and different functional states. Centrifugal countercurrent distribution (CCCD) analysis can also be used in an aqueous two-phase system to determine the relationship between sperm parameters and in vivo fertility in ewes. In a previous work, we found a significant correlation between two post-CCCD parameters (heterogeneity and recovered viability) and field fertility when the same sample was used after cervical AI. The present study was intended to find out whether the control of several external factors that affect reproductive efficiency is able to increase the correlation coefficient between post-CCCD parameters and fertility. Thus, 90 Rasa aragonesa ewes were controlled on the same farm and received intrauterine inseminations using the same technical equipment. The fertilizing ability of the raw semen and sperm samples selected by a dextran/swim-up process was compared using a low number of spermatozoa per insemination (7 × 10 7) to enhance possible fertility differences. A new post-CCCD parameter was considered; the loss of viability (LV) occurred during the CCCD process. This variable denotes the sperm surviving ability and corresponds to the difference between the total number of viable cells loaded and recovered after the CCCD run. The mean fertility of eight sperm control samples was 60% (range: 25–76%), and there was no significant correlation between standard parameters and in vivo fertility. LV ranged from 2 to 69% (average 27%) and was negatively correlated with fertility ( r = −0.914, P < 0.01). Ejaculate heterogeneity (H) ranged from 20 to 47% and was positively, but not significantly, correlated with fertility ( r = 0.391). A predictive equation for fertility was deduced by multiple analysis with a very high correlation coefficient ( r = 0.967), and level of significance ( P < 0.005): predictive fertility PF = 52.546 − 0.594 LV + 0.665 H. The mean fertility of 13 swim-up selected samples was 63% (range: 25–86%). Again, only parameters derived from the CCCD analysis were highly correlated with fertility, especially LV and H ( P < 0.05).

Role of cholesterol, calcium, and mitochondrial activity in the susceptibility for cryodamage after a cycle of freezing and thawing

Objective To correlate the levels of two concrete sperm markers, cholesterol and Ca +2, together with mitochondrial activity on raw semen samples with the post-thaw recovery of spermatozoa with progressive motility on human sperm samples as the first step to improve sperm cryostorage protocols. Design Controlled prospective research project. Setting Private and university-affiliated setting. Patient(s) Semen samples from 122 males attending our center for infertility (n = 47) or semen donation (n = 75) were studied. Intervention(s) The mean basic semen parameters of the 122 semen samples studied before and after the freezing and thawing process. Main outcome measure(s) We determined Ca +2 and cholesterol concentrations on seminal plasma by enzymoimmunoanalysis techniques, intracellular Ca +2 concentrations, cholesterol concentrations in the sperm plasma membrane and mitochondrial activity by fluorometry. Result(s) Cholesterol concentration in seminal plasma and cholesterol contents in the sperm membrane and mitochondrial activity were studied. No correlations were initially found to be of statistical significance. Regarding seminal plasma and intracellular sperm Ca +2 concentrations, a statistically significant negative correlation was found ( P=.036 and P=.016). Conclusion(s) Higher cholesterol contents do not appear to protect sperm against cryodamage. Conversely, Ca +2 equilibrium appears to be essential for a good post-thaw recovery. Mitochondrial activity is not reflecting the possibilities of sperm survival and is probably not a good indicator of the sperm metabolism.

Analytical sensitivity of assays used for detection of bovine viral diarrhea virus in semen samples from the Southeastern United States

Bovine viral diarrhea virus (BVDV) is a significant pathogen that can be shed in the semen of infected bulls. Thus, screening for BVDV in semen of bulls is recommended prior to their entry into an artificial insemination center. No previous research has compared the analytical sensitivity of reverse transcription-nested polymerase chain reaction (RT-nPCR) and virus isolation assays for detection of BVDV in semen from an infected bull. Therefore, the goals of this research were to compare the analytical sensitivity of RT-nPCR and virus isolation assays for BVDV in semen and to apply these assays to determine the prevalence in the Southeastern United States of bulls that lack viremia yet shed BVDV in semen. Semen collected from a bull that was persistently infected with BVDV was serially diluted (1/10) in semen from uninfected bulls and frozen in liquid nitrogen as raw, partially extended or fully extended semen. Subsequently, samples of semen were assayed by virus isolation and RT-nPCR. Viral detection was more sensitive in extended semen samples than in raw semen samples and more sensitive by RT-nPCR than virus isolation. After this evaluation of analytical sensitivity, serum and semen were collected from 558 post-pubertal bulls in our region. These samples were tested for BVDV by virus isolation. Partially extended semen was also assayed for BVDV by RT-nPCR. All samples were negative by all assays for BVDV. The application of analytically sensitive assays reveals a very low prevalence (≤0.54%) of BVDV in semen from bulls in the Southeastern United States.

A rapid and sensitive PCR-based diagnostic assay to detect bovine herpesvirus 1 in routine diagnostic submissions

We describe a rapid, sensitive and specific polymerase chain reaction (PCR) assay for the detection of BHV1 DNA in a range of routine diagnostic submissions without the need for prior virus isolation. The assay, which is based on the selected amplification of a portion of the viral tk gene, detected both BHV1.1 and BHV1.2 subtypes in a panel of 15 characterised field isolates, and its sensitivity was estimated to be <0.125 TCID 50. BHV2, alcephaline herpesvirus, BHV4, equine herpesvirus 1 (EHV1), EHV4 and pseudorabies virus were not detected confirming the specificity of the assay. One hundred and five diagnostic submissions, including tissues, nasal secretions and nasal swabs were taken from cattle with respiratory disease and tested using the routine methods of virus isolation (VI) and the fluorescent antibody test (FAT), and the results were compared with those obtained by PCR. The PCR assay detected BHV1 DNA in all samples that were positive by VI. BHV1 DNA was also detectable by PCR in raw and extended semen samples at a sensitivity of 1 TCID 50 per 50 μl. The assay also detected BHV5, permitting differentiation between it and BHV1 by virtue of the size of the amplified PCR product. The PCR assay is more sensitive and independent of sample quality than either virus isolation or FAT, and it is faster than virus isolation. The sample preparation method is simple with few steps involved. There are no extra post-amplification blotting/hybridisation steps and the assay is not based on a nested PCR strategy that might otherwise exacerbate the problem of oversensitivity/contamination in the routine use of such a test in a diagnostic laboratory. This assay would permit discrimination between those animals naturally infected with wild type BHV1 and those vaccinated with tk-BHV1 strains.

Preliminary description of a polymerase chain reaction test for bluetongue and epizootic hemorrhagic disease viral RNA in bovine semen.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are arthropod-borne orbiviruses of worldwide agricultural and economical importance. These viruses are transmitted from animal to animal by biting midges of the genus Culicoides. Several viruses, including BTV, can be isolated in bovine semen. 5 This finding is of considerable concern to the animal breeding industry. The potential presence of BTV and EHDV in bovine semen has resulted in time-consuming and expensive processes for certifying bovine semen free of these viruses. 9 BTV isolation from semen using cell cultures is difficult because of cell culture toxicity of the spermatozoa and seminal plasma. 4 This problem has been circumvented for bovine herpesvirus by the use of polymerase chain reaction (PCR) procedures on bovine semen samples. 12,13 Recently, capture nested PCR procedures were developed that utilize a reverse target capture for viral RNA extraction and reverse transcriptase/nested PCR for BTV and EHDV RNA detection. 11,14 These capture nested PCR tests are BTV and EHDV group specific and can detect all US stereotypes of these viruses. In this study, the capture nested PCR procedure was applied to the detection of BTV or EHDV RNA in virus-spiked raw semen samples. Dilutions of cell culture source viruses were added to virus-free bovine semen and incubated overnight at 4 C. Initially, samples were diluted 2-fold with 10 mM Tris-HCl (pH 8) and sonicated using a cup-horn sonicator for 1 minute. The samples were then microfuged at 12,000 3 g for 10 minutes at 4 C. The pellet was resuspended in 100 ml of sterile water, and 200 ml of extraction buffer 11 was added. The target RNA was denatured and captured using the biotinylated outer antisense primer and strepavidin-coated magnetic beads a as described previously. 11 The sonication and centrifugation step were useful for reducing the protein material, which could cause sample handling problems due to protein aggregates. However, this step was unnecessary for these samples but may be necessary for samples with a higher protein content. In the results reported here, the samples were diluted 2-fold directly with the extraction buffer, and the target RNA was captured as described previously. 11 The captured target RNA was then denatured with 33% formamide and heat, reverse transcribed, and amplified as described previously. 11,14 The products were detected using agarose gel electrophoresis. The BTV assay consistently detected viral RNA from 1‐100 CCID50/0.5 ml and with less consistency down to 0.1 CCID50/0.5 ml (Fig. 1). Samples spiked with 1,000 CCID50/0.5 ml of virus were not detected perhaps because of template inhibition of the PCR amplification. The EHDV assay with spiked raw semen was 100-fold less sensitive (100‐1,000 CCID50/0.5 ml) than

Quality of stallion semen obtained by a new semen collection phantom (Equidame®) versus a missouri® artificial vagina

A study was performed to test a new semen collection device (Equidame® phantom) that fractionates the ejaculate by comparing the quality of semen obtained by the Equidame® phantom with that obtained by a Missouri® artificial vagina. Semen from 4 Finnhorse stallions was collected 4 times per stallion by both methods. Half of the ejaculate was frozen and the other half extended and loaded into 2 Equitainer® transport containers (24- and 48-h samples). Motility parameters were determined by a Hamilton-Thorn motility analyzer after cooled storage for 24 and 48 h and again after freezing/thawing. Raw and chilled semen samples were cultured and the number of bacterial colonies counted after incubations of 24 and 48 h. After a 24-h incubation the number of colony-forming units (CFU) in raw semen was significantly higher (P<0.01) when collected by the Missouri® artificial vagina than by the Equidame® phantom. After cooled storage, 75% of the semen samples contained no bacteria after an incubation of 24 h, and 69% yielded no growth after 48 h. The sperm-rich fractions (Cup 2) collected by the Equidame® phantom had lower mean volumes (22.1 ± 2.3 mL [± SEM] versus 101.6 ± 9.3 mL) and significantly higher mean sperm concentrations (218.0 ± 25.8 × 10 6 vs 86.2 ± 8.1 × 10 6 cells/mL; P<0.05) than the total ejaculates collected by the Missouri® device. The total and progressive motility of chilled and frozen-thawed semen did not differ significantly between collection methods. The Equidame® phantom yielded semen that was of a lower bacteriological colony counts, but had sperm motility similar to that of semen collected with the traditional method by the Missouri® artificial vagina.

Rapid and sensitive detection of the bovine viral diarrhea virus genome in semen.

A reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed for the detection of bovine viral diarrhea virus (BVDV) in cell culture supernatant and in bovine semen. Several sets of primers, PCR conditions and extraction methods were examined to optimize the procedure. A set of primers designed to amplify a highly conserved portion of the p80 gene from BVDV (corresponding to NADL strain sequence from bp 6668 to 7107), was demonstrated to be the most effective. These oligonucleotide primers consistently amplify a 440-bp fragment from several non-cytopathic and cytopathic biotypes of BVDV. The viral origin of the PCR products was assessed by sequencing. The introduction of a Sephacryl S-400 chromatography step to remove seminal inhibitors prior to RNA extraction permitted RT-PCR detection of BVDV in raw and extended semen samples. A maximum sensitivity of 0.4 TCID50 was achieved with this method using RNA extracted from tissue supernatants. This RT-PCR assay may be a useful tool for the detection of BVDV in semen of persistently infected bulls.

Sperm hyperactivation as quality control for sperm penetration assay

The sperm penetration assay (SPA) is subject to considerable variation, and controls are needed to verify the accuracy of the results. It is proposed that sperm hyperaetivation (HA) can serve as a quality control cheek for the SPA. The objective was to determine if there was an association between the SPA outcome and sperm HA measured at various times during the SPA procedure. The data showed a significant correlation between percent sperm HA and percent zona free oocyte penetration by sperm preincubated for three hours prior to sperm-oocyte interaction (short preincubation). Some sperm hyperactivity was observed in liquefied raw semen samples, but this was insignificantly related to SPA results. Low correlation was observed between SPA results and sperm HA determined immediately after centrifuge washing of sperm. The results suggest that it is possible to utilize sperm HA measured immediately after the sperm-oocyte interaction period as a quality control check of SPA results.

Comparison of the abifity of two sperm preparation techniques to remove microbes

The ability of a new sperm preparation method--self migration in sodium hyaluronate (SH)--to remove microbes from samples was compared to that of the traditional method of centrifugation/swim up (CS). Two 1-ml aliquots were taken from 20 semen samples used for inseminations, and prepared by each of the methods. Samples for culture were taken immediately before wash and at 2 and 24 h after. Microbes were found in all raw semen samples with a mean concentration of 47.8 x 10(3) colony forming units per ml (c.f.u./ml). After preparation and 2 h incubation with and without penicillin, both CS and SH were found greatly to reduce the number of isolates. There were no differences between the ability of the two methods to reduce microbe concentration, although SH recovered more progressive motile spermatozoa than CS (P less than 0.05). When penicillin preparation was omitted, both the CS and SH methods resulted in significantly more isolates after 24 h of incubation. This effect of penicillin preparation was also evident after only 2 h of incubation with CS, but not with SH. No clinical signs of infection were observed among women receiving treatment. We conclude that sperm preparation by self migration in sodium hyaluronate is a simple and safe method to remove microbes and to recover motile spermatozoa.