Showing papers on "Semen published in 1997"

Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro.

Abstract: The objective of this study was to determine the incidence of DNA fragmentation in human sperm, and to correlate any detected DNA damage with semen analysis parameters and fertilization rates in in vitro fertilization (IVF). A total of 298 semen samples were collected from men in the infertility program at The Toronto Hospital. For each sample, the percentage of sperm with DNA fragmentation was determined using the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) and fluorescence-activated cell sorting. The percentage of sperm with fragmented DNA was less than 4% in the majority of samples but ranged from 5% to 40% in approximately 27% of the samples. A negative correlation was found between the percentage of DNA fragmentation and the motility, morphology, and concentration of the ejaculated sperm. In 143 IVF samples, a significant negative association was also found between the percentage of sperm with DNA fragmentation and fertilization rate (p = 0.008) and embryo cleavage rate (p = 0.01). In addition, 35 men who smoked demonstrated an increased percentage of sperm with fragmented DNA (4.7 +/- 1.2%) as compared to 78 nonsmokers (1.1 +/- 0.2%; p = 0.01). These results demonstrate a negative association between semen analysis parameters and sperm with fragmented DNA. Since extremely poor semen samples are the indication for intracytoplasmic sperm injection, there is a high likelihood that sperm with fragmented DNA may be selected by chance and used for oocyte injection, resulting in poor fertilization and/or cleavage rates.

Objectively measured boar sperm motility parameters correlate with the outcomes of on-farm inseminations: results of two fertility trials.

Abstract: Two fertility trials were undertaken to evaluate the relationship between boar semen quality and fertility (conception rate and litter size) after on-farm artificial insemination (AI). Trial 1 included 98 ejaculates from 27 boars, and trial 2 included 72 ejaculates from 26 boars. The semen quality was measured by computer-assisted semen analysis (CASA) using the Hobson Sperm Tracker. Boar semen was diluted in a standard extender (Beltsville Thawing Solution, BTS), dispensed into 75 ml allquots each containing 1.5 x 10(9) spermatozoa and dispatched to farms by overnight mail for use by their normal AI procedures. Randomly selected 75 ml aliquots of semen from each boar were also sent to the institute of Zoology for CASA measurement. Prior to CASA analysis, the spermatozoa were recovered from the BTS using Percoll gradients, resuspended in trisbuffered saline media containing 40 mM Ca++, and incubated at 39 degrees C. Parameters of sperm motion were measured after 0, 2, 4, and 6 hours of incubation. Various multiple regression models based on measured motion parameters could account for up to 24% of the variation in litter size. Using logistic regression, highly significant (P < 0.0001) models explaining conception rate in terms of sperm motion were derived for trial 2 only. The change in sperm velocity during the first 2 hours of incubation and the magnitude of the velocity parameters after 2 hours were identified as the most consistent indicators of fertility. Other attributes of sperm quality, i.e., frequency of spontaneous acrosome reactions (AR) and ARs induced by ionophore A23187 or solubilized pig zona pellucida, were also examined. When the "within-trial" median litter size was used as a way of allocating ejaculates to "high" or "low" litter-size groups, higher litter size was associated with lower frequency of both spontaneous and induced AR. These results demonstrate that fertility information can be derived from the CASA analysis of boar semen provided it is combined with a period of incubation in capacitating conditions.

High viral load in semen of human immunodeficiency virus type 1-infected men at all stages of disease and its reduction by therapy with protease and nonnucleoside reverse transcriptase inhibitors.

Abstract: Seminal viral load is likely to be directly related to the sexual transmissibility of human immunodeficiency virus type 1 (HIV-1). However, it is not clear whether the level of HIV-1 in semen varies with the stage of infection and whether antiretroviral therapy reduces seminal viral load. A nucleic acid sequence-based amplification (NASBA) technique was used to quantify HIV-1 RNA as an indicator of infectious viral load in semen and blood plasma of homosexual men with different stages and durations of HIV-1 infection. The median viral load in a cross section of 34 men was 11,000 HIV-1 RNA copies/ml (range, <400 to 1.3 x 10(7) copies/ml) in whole semen and 5,238 HIV-1 RNA copies/ml (range, <400 to 2.8 x 10(5) copies/ml) in seminal plasma, which is 10- to 1,000-fold higher than previous estimates. Viral loads in whole semen and seminal plasma were strongly correlated with blood plasma viral load (P < 0.001) but not with blood CD4+ T-cell count (P = 0.420). Longitudinal analysis of eight subjects who progressed to AIDS showed that seminal viral load increased in most cases, with viral load consistently higher in blood plasma than in semen. Viral loads in semen and blood plasma decreased markedly in six other patients following initiation of potent combination therapy with a protease inhibitor (indinavir) and a nonnucleoside reverse transcriptase inhibitor (DMP-266). These findings have important implications for the biology of sexual transmission of HIV-1 and its potential reduction by antiretroviral therapy.

T Lymphocytes and Macrophages, but Not Motile Spermatozoa, Are a Significant Source of Human Immunodeficiency Virus in Semen

Abstract: The cellular fraction of semen contains spermatozoa, immature germ cells, leukocytes, and epithelial cells Recent evidence implicates seminal cells as a major source of sexually transmitted human immunodeficiency virus (HIV) in semen, but the identity and infectious potential of infected cells remains poorly understood HIV provirus was found in 75% of viable semen cell samples by polymerase chain reaction and in 88% of paired blood cell samples from HIV-seropositive men When semen cell subpopulations were isolated by an immunomagnetic bead technique, T cells were found to be most commonly HIV-infected (75% of samples), followed by macrophages (38% of samples) Viral DNA was never detected in motile spermatozoa or immature germ cell populations Semen leukocytes proliferated in response to mitogenic and antigenic challenge and produced p24 following stimulation with irradiated allogeneic cells These data provide evidence that both T cells and macrophages, but not germ cells, are cellular vectors of HIV transmission in semen

Premature Capacitation of Bovine Spermatozoa Is Initiated by Cryopreservation

Abstract: Poor motility and abnormal acrosomal morphology only partially explain the reduced fertility of cryopreserved bovine spermatozoa. To test the hypothesis that cryopreservation procedures (dilution, cooling, freeze-thaw) induce capacitation in bovine spermatozoa, two experiments were conducted using semen diluted in egg yolk-Tris-glycerol extender (EYTG) (Tris, tris(hydroxymethyl)aminomethane). Capacitation was determined prior to and following incubation with various concentrations of heparin using the chlortetracycline (CTC) fluorescence assay or after preexposure to EYTG using in vitro fertilization (IVF) of bovine cumulus-oocyte complexes (COC) in the absence of heparin. Fresh ejaculates were divided into four treatments and the first was diluted with noncapacitating medium, NCM (+0.3% polyvinyl alcohol (PVA); control), then maintained at 23 degrees C for 4 hours. The remaining semen was diluted with EYTG; the second treatment was held at 4 degrees C (EYTG-4), and the third treatment was held at 23 degrees C (EYTG-23) for 4 hours. The fourth treatment was cooled to 4 degrees C over 4 hours, as per the normal industry protocol, cryopreserved, and thawed (frozen-thawed). After the 4-hour maintenance periods or thawing, all treatments were resuspended either in capacitating medium (CM; +0.6% BSA) for the CTC experiment (n = 3) or in NCM for the IVF experiment (n = 9-11). Prior to incubation in conditions that support capacitation, the percentage of cells exhibiting pattern B (capacitated according to the CTC assay) was similar for all treatments with fresh-extended spermatozoa. Immediately following the addition of heparin (0, 2, or 10 micrograms/ml), three times more frozen-thawed than fresh-extended spermatozoa exhibited pattern B (P < 0.05). After 3 or 6 hours of incubation, however, the percentages of cells displaying pattern B did not differ among treatments. In the absence of heparin, spermatozoa preexposed to EYTG-4 fertilized 2.6x more COC than did control cells (P < 0.001) and 9.2x more than spermatozoa preexposed to EYTG but held at 23 degrees C (EYTG-23; P < 0.0001). No differences were observed among fertilization rates for fresh-extended (EYTG-4) and frozen-thawed spermatozoa. This study provides evidence that premature capacitation occurs in partially (extended and cooled) and fully cryopreserved bovine spermatozoa.

Chlortetracycline analysis of boar spermatozoa after incubation, flow cytometric sorting, cooling, or cryopreservation.

Abstract: In this study, the effects of staining procedure with chlortetracycline (CTC) and method of analysis of boar spermatozoa after staining were examined. The hypothesis that incubation, flow cytometric sorting, cooling, and cryopreservation cause changes to boar sperm membranes which resemble capacitation and the acrosome reaction was also tested. Membrane status was evaluated by flow cytometry and by fluorescence microscopy after staining with CTC, and acrosome integrity was checked by flow cytometry after staining with FITC-pisum sativum agglutenin and propidium iodide (PI). Flow cytometry was also used to assess viability (percentages of live and dead cells) of boar sperm after staining with SYBR-14 and PI. Staining of spermatozoa with CTC alone and in combination with PI and/or Hoechst 33342 had no effect on the proportion of spermatozoa allocated to the F (uncapacitated), B (capacitated), or AR (acrosome-reacted) CTC fluorescent staining categories. The mean percentages of acrosome-intact and acrosome-reacted cells were 88.4 and 6.8 or 0.8 and 96.5 in semen treated with 0 or 100 microg/ml lysophosphatidylchloine (LPC), respectively (P < 0.001). Most spermatozoa were also in the AR CTC-stained category after treatment with LPC compared with a small proportion in the controls. Using flow cytometry to examine sperm suspensions stained with CTC, a gated population of spermatozoa with low fluorescence (population 1) comprised predominantly F-pattern cells (F-pattern: population 1 vs. population 2, 80.5 vs. 14.4%; P < 0.001), whereas population 2 (high fluorescence) comprised mainly B-pattern cells (B-pattern: population 1 vs. population 2, 8.5 vs. 62.3%; P < 0.001). Incubation (38 degrees C, 4 hr), flow sorting, cooling (to 15 or 5 degrees C) and freezing reduced the proportion of F-pattern and live spermatozoa, and increased the proportion of B-, AR-pattern, and dead spermatozoa, in comparison with fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C (30.4, 48.5, 21.1%) than in those cooled to 15 degrees C (56.1, 32.6, 11.5% F-, B-, and AR-pattern spermatozoa, respectively).

Semen parameters in a fertile versus subfertile population: a need for change in the interpretation of semen testing

Abstract: This prospectively designed study was conducted to compare a fertile and a subfertile population so as to define normal values for different semen parameters. Semen analyses were performed according to the World Health Organization (WHO) guidelines, except for sperm morphology (strict criteria). In the fertile population (n = 144), all patients had recently achieved pregnancy, within 12 months of unprotected coitus. As subfertile controls we examined semen samples from 143 consecutive men attending our infertility clinic during the same study period. Couples with tubal factor infertility and/or ovulatory disorders were excluded from our study. Using receiver operating characteristic (ROC) curve analysis we determined the diagnostic potential and cut-off values for single and combined sperm parameters. Sperm morphology scored best, with a value of 78% (area under the ROC curve). Summary statistics showed a shift towards abnormality for most semen parameters in the subfertile population. Using the 10th percentile of the fertile population as the cut-off value, the following results were obtained: 14.3 x 10(6)/ml for sperm concentration, 28% for progressive motility and 5% for sperm morphology. Using ROC analysis, cut-off values were 34 x 10(6)/ml, 45% and 10% respectively. Cut-off values for normality were different from those described in the WHO guidelines. Routine bacterial and non-bacterial cultures turned out to be of little prognostic value.

Quantification of HIV in semen: correlation with antiviral treatment and immune status

Abstract: Objective: This study examined the concentration of HIV in semen and the effects of biological factors on HIV excretion Methods: Semen samples from 101 men at different stages of the disease were evaluated by quantitative HIV culture and HIV RNA detection Blood plasma samples were available from 56 patients The effects of CD4 and CD8 count, blood plasma RNA levels, treatment status and clinical staging on the shedding of HIV were evaluated Results: HIV RNA levels in semen correlated with quantitative HIV culture of seminal cells and a strong association of positive seminal cell culture with high RNA levels was observed CD4 count and antiviral treatment were inversely correlated with the concentration of HIV in semen Blood plasma HIV RNA values were correlated with HIV RNA levels in semen, although some patients had highly discrepant results Conclusions: The strong correlation between seminal cell culture and concentration of HIV RNA in seminal plasma suggested that HIV detected in seminal plasma was released by productively infected cells in the male genital tract The study showed that the concentration of HIV in semen, which was likely to be correlated with HIV infectivity, was a function of the immune status of the HIV-infected individual The results suggested that antiviral therapy may have reduced the concentration of HIV in semen

Effect of antiviral treatment on the shedding of HIV-1 in semen.

Abstract: Objective: The potential role of antiretroviral treatment on the infectiousness of HIV1-infected men was examined by studying the effect of antiviral treatment on the shedding of HIV-1 in semen. Methods: Forty-four patients enrolled in various treatment protocols were asked to donate a semen sample before they began a new antiviral treatment and at a followup visit after 6 to 15 weeks of treatment. Since most patients were on blinded protocols, patients were stratified by response of blood viral load. The effect of each patient’s treatment was classified as good (n = 24), fair (n = 8) and marginal (n = 13) by measurement of the HIV RNA reduction in blood plasma (> 1.0 log10; 0.5‐1.0 log 10 and <0.5 log 10 HIV RNA copies/ml reduction, respectively). The effect of treatment on shedding of HIV-1 in semen was documented by the reduction of HIV RNA concentration in seminal plasma and by quantitative HIV-1 seminal cell culture. Results: Overall, antiviral treatment resulted in a significant fall in the viral load in semen (RNA and culture) that paralleled the reduction of viral load in blood. More pronounced reductions of HIV RNA in semen were observed as the effectiveness of treatment on blood HIV RNA levels increased (median drop from baseline 0, 0.3 log 10 and 0.8 log 10 RNA copies/ml in patients with marginal, fair and good treatment effect, respectively). Thirteen patients lost detectable HIV RNA in blood on treatment and all of these had undetectable levels of HIV-1 in semen by culture and RNA analysis at follow-up. In 19 of the 31 patients (62%) who still had HIV RNA in their blood during treatment, semen HIV levels were below detection in semen at follow-up. Conclusions: Treatment-induced changes of HIV RNA concentration in blood are generally associated with a corresponding change in seminal HIV RNA. If confirmed in larger studies, potent antiretroviral therapy might reduce the spread of HIV-1.

Effects of dietary selenium and vitamin E on boar performance and tissue responses, semen quality, and subsequent fertilization rates in mature gilts.

Abstract: Three experiments involving 192 crossbred boars evaluated the effects of dietary Se (0 or .5 ppm) and vitamin E (0 or 220 IU/kg) on growth, tissue Se, and alpha-tocopherol concentrations, and on semen quality and its subsequent effect on fertilization rate in mature gilts. Diets formulated used torula yeast and dextrose or cornstarch as the basal feedstuffs and were provided from weaning through sexual maturity. The basal diets averaged .063 ppm Se and 3.46 mg alpha-tocopherol/kg diet. Experiment 1 was a 2 x 2 factorial and conducted as a randomized complete block design in six replicates. Boars were allotted at weaning (initial BW 7.7 kg) with growth and feed performance determined to 145 kg BW. Five boars were killed at weaning and three from each treatment group at periodic intervals to 145 kg BW. Serum and tissue Se and alpha-tocopherol concentration and glutathione peroxidase (GSH-Px) activity were subsequently determined. No performance benefit from either nutrient was demonstrated. Tissue (serum, liver, and testis) GSH-Px activity and Se and alpha-tocopherol concentrations were higher (P < .01) at each period when that respective nutrient fortified the diet. Testis GSH-Px activity increased from weaning to 145 kg BW even when Se was not added to the diet. Experiment 2 was conducted after training three boars from each treatment group of Exp. 1 for semen collection. From 9 mo of age and for a 16-wk period, semen was collected three times weekly and the volume, sperm concentration, motility, and percentage of normal and abnormal sperm were determined. Boars fed either the nonfortified Se or vitamin E diets had sperm with lower motilities (P < .01) and a higher percentage of sperm cells with bent and shoehook tails (P < .01). Diets low in added Se seemed to have a greater detrimental effect on the percentage of motile and abnormal sperm than diets inadequate in vitamin E. Sperm cells had a high concentration of Se and alpha-tocopherol, and a high GSH-Px activity. Experiment 3 was conducted using the boars from Exp. 2; 34 mature gilts were inseminated at 12 and 24 h after estrus. Gilts were killed 5 to 7 d postcoitum and the reproductive tracts were recovered. The semen from boars fed the nonfortified Se diet had a lower fertilization rate of oocytes with fewer accessory sperm penetrating the zona pellucida. The results from these experiments indicate that dietary Se and vitamin E can affect boar semen quality, but the greater effect seemed to be from Se.

Lipid and antioxidant changes in spermatozoa and seminal plasma throughout the reproductive period of bulls

Abstract: The lipid compositions and associated antioxidant capacities of spermatozoa and seminal plasma from bulls were examined at the beginning, middle and end of their reproductive period. The reduction in concentration and motility of spermatozoa associated with ageing was accompanied by a large decrease in lipid concentrations within the seminal plasma; this change in lipid concentration was accompanied by an increase in the proportion of phospholipid. By contrast, the proportion of phospholipids in the spermatozoa was significantly reduced. The major phospholipid fractions within both the spermatozoa and seminal plasma were phosphatidyl choline and phosphatidyl ethanolamine. With increasing age there was a large decrease in the proportion of phosphatidyl ethanolamine and a commensurate increase in that of phosphatidyl choline within the spermatozoa and seminal plasma. These major changes in phospholipids were accompanied by a decrease in the amount of phosphatidyl inositol and an increase in that of cardiolipin in both spermatozoa and seminal plasma. The reductions in the proportions of phosphatidyl ethanolamine were accompanied by extensive reductions in the content of the major polyunsaturated fatty acids, arachidonic 20:4 (n-6) and docosahexaenoic 22:6 (n-3); there was a decrease also in the concentration of 22:6 (n-3) in phosphatidyl choline. The changes in lipid composition owing to ageing were associated with a marked reduction within the seminal plasma of the major antioxidant enzyme systems, glutathione peroxidase and superoxide dismutase.

In vivo semen-associated pH neutralization of cervicovaginal secretions.

Abstract: Physiological cervicovaginal acidity can partly inactivate human immunodeficiency virus (HIV). Basic semen components should be able to partially neutralize in vivo cervicovaginal pH. The goals of the study were to evaluate the relationship between cervicovaginal pH and presence of semen components in sexually active African women and to assess whether vaginal douching with water performed just after sexual intercourse could significantly reduce semen components and restore physiological cervicovaginal pH. Cervicovaginal secretion (CVS) from 56 heterosexual African women (19 to 45 years old), living in Bangui, Central African Republic, were evaluated for pH, semen components (prostatic acid phosphatase [PAP] and prostatic specific antigen [PSA]), cellularity, and hemoglobin at inclusion and after vaginal douching with 100 ml of water by using a bock. Before douching, semen components were found in 46 of 56 CVS (82%). The mean vaginal pH was 5.2 (range, 3.6 to 7.7), and concentrations of both PAP and PSA correlated positively and strongly with cervicovaginal pH (P < 0.001). After douching, semen components were found in 35 of 56 CVS (62%) (P = 0.03). Cervicovaginal PAP and PSA levels were significantly decreased (respectively, P < 0.0001 and P < 0.01; PAP, -72%; PSA, -87%), as was the total cell count (-60%; P < 0.0001). Furthermore, in CVS previously positive for both PAP and PSA, the mean vaginal pH was significantly decreased (6.5 versus 5.3, P < 0.01); no genital bleeding was observed. Frequent persistence of semen in CVS from heterosexually active African women leads to a shift from acidity to neutrality that could favor male to female HIV transmission. Vaginal douching provides significant elimination of semen after sexual intercourse; it should be considered for study as a supplementary means for the prevention of heterosexual HIV transmission.

Detection of human herpesvirus 8 DNA in semen from HIV-infected individuals but not healthy semen donors.

Abstract: Objective: To ascertain the prevalence of Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus (HHV) type 8, and cytomegalovirus (CMV) DNA in semen was investigated.Methods: Amplification by nested polymerase chain reaction was used to detect viral DNA sequences in samples from 24 HIV-infected gay men, 15 of them with Kaposi's sarcoma (KS), and 115 healthy donors.Results: Six of the 24 HIV-infected patients had detectable HHV-8 DNA in their semen: three of the 15 patients with KS and three of the nine patients without KS. CMV DNA was detected in 20 semen samples from HIV-infected patients. None of the semen samples from healthy donors had detectable HHV-8 DNA and rates of CMV DNA detection were tow (3%).Conclusions: The study demonstrates the presence of HHV-8 in semen from HIV-infected individuals with, or at risk, of developing KS and the potential for sexual transmission of the virus. We found no evidence of HHV-8 in the semen of HIV-uninfected donors.

Effect of dietary fat on the fatty acid composition and fertilizing ability of fowl semen.

Abstract: Broiler breeder roosters received two diets, containing either 5% salmon oil (SO) or 5% corn oil (CO). The diets differed essentially in their polyunsaturated fatty acid (PUFA) composition, with n-6:n-3 fatty acid ratios of 41.6 in SO and 1.5 in CO. The effects of these diets on the fatty acid composition of spermatozoa and seminal plasma, and on fertility evaluated after artificial insemination were observed. Whatever the diet, the fatty acid composition of spermatozoa showed notable amounts of 20:4n-6 (5-9%) and 22:4n-6 (15-21%). These essential fatty acids were not detected in the diets and were synthesized from 18:2n-6, which was abundant in the diet (15-16%) but low in spermatozoa (2-3%). Spermatozoa were also very rich in saturated fatty acids (39%). There was a clear influence of dietary lipids on the spermatozoa fatty acid profile: the proportion of n-3 fatty acids in spermatozoa from males fed SO compared to CO was higher (9.6% vs. 4.3%) and that of n-6 fatty acids was lower (22.4% vs. 33.3%). The fatty acid composition of seminal plasma included a higher proportion of saturated fatty acids (49%) than the proportion in spermatozoa, whereas minor fatty acids (14:0, 16:1n-7, 16:1n-9, 22:5n-3) were not detected. The influence of dietary lipids on the seminal plasma fatty acid profile was the same as for the spermatozoa, especially in the PUFA profile. In addition, the SO diet gave significantly higher fertility rates (96%) than the CO diet (91.6%). These results clearly show that the lipid composition of the diet may modify the fatty acid composition of the semen and its fertilizing ability.

Evidence for regional differences of semen quality among fertile French men. Fédération Francaise des Centres d'Etude et de Conservation des Oeufs et du Sperme humains.

Abstract: The world literature on human semen quality indicates apparent geographical differences but these might primarily depend on variations among studies for subject recruitment strategy, semen analysis or data processing methods. A retrospective analysis on the quality of semen from 4710 healthy unselected fertile men, who were candidate semen donors to sperm banks in university hospitals in eight different French areas during the period 1973-1993, was undertaken. In these centres, all the men were referred under the same guidelines and all semen samples were analysed using similar methodologies. Significant differences were found between centres for seminal volume, sperm concentration, total number of spermatozoa in the ejaculate and percentage of motile spermatozoa (all P < 0.0001). Multiple regression analysis accounting for the age, sexual abstinence before semen collection and year of semen collection also showed regional differences: compared to Paris, the seminal volume was higher in Caen (P < 0.001) and lower in Toulouse (P < 0.01), the total number of spermatozoa was higher in Lille (P < 0.001) and lower in Toulouse (P < 0.05) and the percentage of motile spermatozoa was higher in Bordeaux and lower in Tours (both P < 0.001). This is the first study providing evidence for regional differences in the human semen quality.

Methanol as cryoprotectant and the suitability of 1.2 ml and 5 ml straws for cryopreservation of semen from salmonid fishes

Abstract: For salmonid semen, the cryoprotective action of 10% methanol was compared with a 5% dimethyl sulphoxide (DMSO), 1% glycerol mixture, until now one of the most effective cryoprotectants. In Oncorhynchus mykiss (Walbaum), Salmo trutta L. f. fario, Salmo trutta L. f. lacustris and Salvelinus alpinus (L.), semen cryopreserved with both cryoprotectants yielded post-thaw fertilization rates of 90-100% of control with untreated semen at sperm-to-egg ratios of 1.8 × 106-2.4 × 106 spermatozoa per egg. However, at sperm-to-egg ratios of 0.9 × 106-1.2 × 106 spermatozoa per egg, semen cryopreserved with methanol had significantly higher fertilization rates than semen frozen with the DMSO/glycerol mixture. In other studies we obtained similar data for Coregonus sp., Salvelinus fontinalis (Mitchill), Thymallus thymallus (L.) and Hucho hucho (L.), proving that methanol is the most effective and generally applicable cryoprotectant for semen of the studied salmonid species. To facilitate the insemination of large egg batches we investigated the suitability of 1.2 ml and 5 ml straws for deep freezing of semen of Oncorhynchus mykiss, Salmo trutta f. fario, Salmo trutta f. lacustris and Salvelinus alpinus. With 1.2 ml straws the fertilization rates were similar to 0.5 ml straws when using lower freezing and higher thawing temperatures. The 5 ml straws resulted in a fertilization success of only about 40% of fresh semen control.

Fertilization and pregnancy using intentionally cryopreserved testicular tissue as the sperm source for intracytoplasmic sperm injection in 10 men with non-obstructive azoospermia.

Abstract: Testicular tissue extraction (TESE) to obtain spermatozoa for use with intracytoplasmic sperm injection (ICSI) has recently been employed in patients with non-obstructive azoospermia. Standard protocol is to retrieve a new sample of testis tissue on the day of oocyte recovery. Unfortunately, approximately 30% of men will possess no spermatozoa in their tissue, making ICSI an impossibility. We investigated whether testicular tissue that was intentionally obtained well before any planned ICSI cycle and cryopreserved could then serve as an efficacious sperm source in a subsequent ICSI cycle. This study reports on 10 men with non-obstructive azoospermia who did have spermatozoa found within their testis tissue at the time of TESE and who chose to use their frozen samples as the source of spermatozoa for a later cycle of ICSI. In 19 cycles the overall fertilization rate was 48%. Embryo transfer occurred in 89% of cycles. Two couples have achieved pregnancy (one ongoing, one delivered). All patients except one had multiple vials of frozen tissue remaining following their first cycle. This approach is offered as an alternative to repeated testicular tissue sampling, as the availability of spermatozoa is assured prior to the initiation of ovulation induction. This tissue can be harvested at the same time as diagnostic biopsy, thereby minimizing the number of surgical procedures.

Size differences between human X and Y spermatozoa and prefertilization diagnosis.

Abstract: Normal human spermatozoa carry either the X or the Y chromosome. The differences between X and Y spermatozoa (X and Y haploid cells) may exist in two areas: the different chromosomes (i.e. different kinds and numbers of genes) and the different sperm structures and functions (i.e. different genetic expression). The aim of this study was to determine whether there are any size between X and Y spermatozoa and whether sperm size and shape varies between men. Identification of the Y (and X inferred) status of individual spermatozoa was carried out by polymerase chain reaction (PCR), amplifying the putative testis-determining gene (SRY) together with a control gene (ZP3). PCR amplification of 871 out of 895 (97.3%) single motile spermatozoa showed that 444 (51.0%) were Y and 427 (49.0%) were X-bearing spermatozoa. Of 233 normally-shaped but immobilized spermatozoa, 217 (93.1%) were photographed and measured. Statistically, the length, perimeter and area of the sperm heads, and the length of the sperm necks and tails of X-bearing spermatozoa were significantly larger and longer than those of Y-bearing spermatozoa. Some peculiarities (or variations) in the X and Y sperm shape and size in individual donors were found. The pre-screening by micro-measurement of these specific haploid characteristics of individual spermatozoa in different donors, which may be closely related to their different genetic conditions (or diseases), may be important in human medicine and animal husbandry, especially in sperm prefertilization diagnosis.

Chromatin structural changes in sperm after scrotal insulation of Holstein bulls.

Abstract: The reported effects on semen quality ascribed to testicular heat stress generally relate to traits impacting sperm transport and fertilizing ability but not to the genetic material contained by the sperm. To characterize the effects of testicular heat stress on sperm chromatin, susceptibility of DNA in sperm nuclear chromatin to in situ acid denaturation was measured by flow cytometry after staining with acridine orange using the sperm chromatin structure assay (SCSA). Semen was collected from Holstein bulls at 3-day intervals, before and after 48-hour scrotal insulation, until the morphologically abnormal sperm content in raw semen exceeded 50%. After cryopreservation in egg yolk-citrate extender, semen was thawed and sampled during incubation in vitro at 38.5 degrees C. Overall, SCSA results showed that chromatin susceptibility to denaturation was increased for sperm collected post- vs. preinsulation and was more pronounced for sperm presumably in the testes during insulation than for those sperm presumably in the epididymides. Increased susceptibility was detected as early as the first collection postinsulation; however, chromatin of sperm presumably in the proximal epididymis during insulation did not appear to have been detrimentally affected. Chromatin susceptibility to denaturation increased with increased incubation time in vitro, but the rate of change in susceptibility during incubation did not differ among pre- vs. postinsulation specimens. We conclude that elevated scrotal temperatures adversely affect both epididymal and testicular sperm by reducing sperm chromatin stability. The effects of heat stress on the chromatin of epididymal sperm were more subtle than those exhibited by testicular sperm but detectable within close proximity to the heat stress event.