Ejaculate Concentration Research Articles

The Effects of Adding Hempseed Cake on Sperm Traits, Body Weight, Haematological and Biochemical Parameters in Rabbit Males.

Incorporating of agro-industrial co-products into animal nutrition could represent an opportunity to lessen the environmental impact of the food production chain. One such co-product is a hempseed cake originating from cold pressing hemp seeds to extract oil for human consumption. The aim of the present study was to evaluate the action of hempseed cake in the diet on male rabbit reproductive and some non-reproductive indexes. Male rabbits were fed either a standard diet (control group; C; n = 10) or a diet enriched with hempseed cake (experimental group E5 with 5% of a hempseed cake; n = 10, and experimental group E10 with 10% of a hempseed cake; n = 10) in 100 kg of the milled complete feed mixture. Rabbit weight gain, sperm concentration, motility, progressive motility, and sperm quality were evaluated using CASA and flow cytometry. Feeding with a hempseed cake, given at both tested concentrations, had no effect on weight gain per week and the total average weight gain compared to the control group (p > 0.05). Hempseed cake addition had no effect on sperm concentration in ejaculate, sperm motility, and progressive motility (p > 0.05). Selected haematological and biochemical indexes were examined. The E5 group showed positive tendencies in hepatic profile parameters, while in the E10 group the tendencies were opposite, though within the reference values. Based on our results, no negative effects of hempseed cake feeding on rabbit reproduction and health status were found, and we can recommend the use of hempseed cake at doses up to 10% in the nutrition and feeding of rabbits. Therefore, agro-industrial co-products can decrease the feeding cost.

INFLUENCE OF THE STRUCTURE OF DIETS ON THE PRODUCTIVITY OF SERVICING BULLS

Complete feeding of young replacement and adult breeding animals makes it possible to provide the diets of animals with the necessary nutrients, which have a positive effect on the semen quality and, consequently, increase the period of economic use. Therefore, the purpose of the study was to optimize the structure of diets for servicing bulls. It was found that feeding the experimental diets of winter and summer periods with the same type of feeding increased the ejaculate volume by 4.0-4.2%, sperm activity - by 3.1-6.4%, sperm concentration in ejaculate - by 13.8-15.3%.

The effect of preparatory therapy with myo-inositol on the outcome of infertility treatment by ART in men with low reproductive function

Research objective: to study the effect of myo-inositol (MI), folic acid (FA) and cyanocobalamin in mono- and complex therapy on the classical fertility rates and the level of DNA fragmentation in sperm in men with low reproductive function, and on the results of the in vitro fertilization (IVF) programs performed in these patients.Materials and methods. A retrospective study of 133 patients aged 32 to 40 years was carried out for the period 2020–2021. All patients were divided into three groups: group 1 – 44 men who took MI, FA and cyanocobalamin daily as monotherapy for 3 months; group 2 – 42 patients who followed a diet to improve spermatogenesis for 3 months; group 3 – 47 men, for whom complex therapy was applied for 3 months: MI, FA, cyanocobalamin and diet.Results. The increase of sperm concentration in ejaculate, improvement of progressively motility of spermatozoa, and increase of the percent of spermatozoa with normal morphology in men with failures of reproductive function after the complex preparatory therapy are defined. The preparatory therapy for men with MI in preparation for IVF helps to improve the quality of embryos and increase the pregnancy rates in couples with male infertility due to the assisted reproductive technologies. The positive effect of preparatory complex therapy in patients with poor spermatogenesis on the blastocyst formation rates in IVF programs is shown.Conclusions. The positive effect of MI on the normal functioning of the reproductive system in men has been proven. The increase pregnancy rates in the IVF program after preparatory treatment of men with MI can be explained by its positive effect on reducing the level of DNA fragmentation of sperm.

P–053 Concentration of bisphenol S in human seminal plasma negatively correlates with decreasing sperm concentration in ejaculate

Abstract Study question Is there a relationship between concentration of bisphenol S in seminal fluid and spermiogram parameters? Summary answer Bisphenol S was detected in 81% of seminal plasma samples. Negative correlation was found between BPS concentration and total sperm count in normozoospermic men. What is known already Human spermatogenesis can be influenced by a range of chemicals present in our environment. Bisphenol S (BPS) is a very frequent compound commonly used as a softener in production of plastics, where it has replaced bisphenol A. It is an endocrine disruptor frequently associated with negative effects on reproduction. It has been observed that BPS can affect testicular development in rodent males. In addition, it has cytotoxic, reprotoxic and neurotoxic effects and induces the oxidative stress bringing negative effects on spermatogenesis. BPS has been detected in food, drinks or cosmetics. Its direct effect on spermatozoa or spermatogenesis is still unclear. Study design, size, duration A total of 38 patients (25 normozoospermic) aged 24 to 42 years, non-smokers, with BMI between 19,9–32,9 were included in this prospective study from 2018 to 2020. None of them had varicocele, urogenital infections or other urological problems. Their seminal plasma was separated by centrifugation. BPS was extracted using solvent extraction followed by preconcentration step. The samples were analysed on Agilent 6495 Triple Quadrupole. Two MS/MS transitions were used for quantitative LC-MS/MS analyses. Participants/materials, setting, methods All the men included in this study signed an informed consent and agreed with analyses of their samples. These analyses were approved by Ethical committee of University Hospital Brno. We evaluated the relationship between concentration of BPS in seminal plasma, sperm concentration, total sperm count, total motility, progressive motility, morphology and fragmentation of DNA in spermatozoa. Statistical evaluation was performed by one individual one-dimensional regression model (p-value lower than 0.05 were considered as statistically significant). Main results and the role of chance The examination revealed the presence of BPS in 31 samples of seminal plasma (81% of all the samples). In 6 samples, the concentration was under level of detection and in one sample under level of quantification. In 7 samples, a very high concentration was detected (>0.1 ng/ml). These values were then compared to spermiogram parameters and sperm DNA integrity. There were no significant differences between the concentration of BPS and morphology of spermatozoa, progressive motility and total motility. In case of the DNA integrity, the opposite trend was observed, lower proportions of spermatozoa with fragmented DNA were found in samples with higher concentrations of BPS. Evaluation of sperm concentration and BPS concentration showed relationship of increasing BPS concentration with significantly lower sperm concentration, the differences were most obvious when only the normozoospermic men were compared. Evaluation of BPS concentration and total sperm count revealed the same trend with statistically significant difference in the category of normozoospermic men. Due to the small number of samples, a negative effect of extreme values on the statistical evaluation cannot be excluded. Currently, more analyses focused on detection of BPS in seminal plasma are carried out in order to obtain sufficiently larger data set. Limitations, reasons for caution A limitation is the number of samples included and analysed in this study, which slightly reduced the power of statistical analysis. Wider implications of the findings: These results document that BPS was present in 81% of analysed samples. Knowing the concentration of BPS in seminal fluid is important for understandig of impact of BPS on male fertility. Our future work will be focused on detection of other bisphenols in seminal plasma. Trial registration number MH CZ – DRO (FNBr, 65269705), AZV NV18–01–00544, Czech Ministry of Education, Youth and Sports (CZ.02.2.69/0.0/0.0/19_074/0012727)

Comparison of sperm parameters and testis histological structure in lake frog (Pelophylax ridibundus) from areas with different level of anthropogenic pollution

The aim. The aim of the work was to study the indicators of spermatogenesis in the lake frog, Pelophylax ridibundus (Pallas, 1771), in the zone of the influence of emissions from the Burshtyn Thermal Power Plant (TPP).
 Materials and methods. Urinal sperm and testes samples were taken from male frogs under conditions of anthropogenic pollution (Burshtyn industrial area). Such parameters as sperm concentration in ejaculate, the number of live spermatozoa, total amount of actively mobile forms, and percentage of spermatozoa with progressive motion were determined by calculations in the hemocytometer. Testis samples were processed for routine histological method. The diameter of seminiferous tubules and histological abnormalities were studied.
 Results. Studies have revealed that near the Burshtyn TPP industrial site, the sperm concentration in the sperm sample and the total number of motile spermatozoa of the lake frogs significantly decreased. The number of dead spermatozoa increases. Amphibians from polluted sites had histological abnormalities in testes such as dilation of the seminiferous tubules with sparse sperm. At the same time, the diameter of the seminiferous tubules in the animals from polluted site significantly decreased.
 Conclusions. The impact of the emissions from the Burshtyn thermal power plant has an expressive reprotoxic effect that is manifested by depressive changes in testes tissue and gametes. The reproductive system of the lake frog is quite sensitive to environmental pollution. Sperm parameters and histological changes in testes are informative biomarkers and can be used for bioindication of technogenically transformed territories

Genome-wide association studies for sperm traits in Assaf sheep breed

Sperm quality traits routinely collected by artificial insemination (AI) center for rams progeny test are related with the capacity to produce sperm doses for AI and, in more or less grade, with males' fertility. Low-quality ejaculates are unuseful to perform AI sperm doses, which suppose high economic loses for the AI center. Moreover, sperm quality traits have low heritability values which make traditional genetic selection little efficient to its improvement. In this work, a genome-wide association study (GWAS) was conducted by using sperm quality traits data and 50 K Affymetrix custom chip genotypes of 429 rams of Assaf breed from OVIGEN AI centre. Furthermore, 47 of these rams were also genotyped with the Illumina HD Ovine BeadChip, and therefore HD genotypes were imputed for all rams with phenotype data. Previous to the GWAS, a linear regression model was fitted including sperm traits as dependent variables; the flock of origin, date of sperm collection, and jump number as fixed effects; rams age at collection in months as covariate; and ram permanent effect as random. Pseudo-phenotypes obtained from this model were used as input for GWAS. Associations at the chromosome-wise level (FDR 10%) of 76 single-nucleotide polymorphisms (SNPs) in 4 chromosomes for ejaculate concentration (CON), 20 SNPs in 3 chromosomes for ejaculate volume (VOL), 32 SNPs in 1 chromosome for ejaculate number of spermatozoa (SPZ), and 23 SNPs for spermatozoa mass motility (MOT) in 17 chromosomes were found. Only SNPs associated with MOT overcame the genome-wide significance level. Some candidate genes for sperm traits variability were SLC9C1 (OAR1), TSN (OAR2), and FUT10 (OAR26) for MOT;. DOCK2, CPLANE1, SPEF2, and RAI14 (OAR16) for CON; SCAPER and PSMA4 (OAR18) for VOL; and PARM1 and LOC101110593 (OAR6) for SPZ. SNPs associated with sperm traits were not found to be correlated with milk production genetic variation; however, the high frequencies of some SNPs with negative effect over sperm traits found in animals at the top milk yield estimated breeding values (EBVs) ranking would allow to exert some selective presure to improve rams sperm performances. Effects and frequencies of some of the SNPs detected over sperm quality traits make these variants good candidates to be used in marker-assisted selection to improve sperm characteristics of Assaf rams and AI center efficiency to produce sperm doses.

Evaluation of Holstein stud bulls by productivity and quality of sperm

The sperm quality of stud bulls is an important indicator on which depends the result of cattle reproduction. The aim of the study was to evaluate the Holstein bulls in terms of productivity and quality of sperm in the conditions of LLC “Ukrainian Genetic Company”. The study was carried out on livestock of 20 stud bulls of the Holstein breed of foreign selection, which were in the same conditions of feeding, keeping and use. The sperm production parameters were determined using an IVOS sperm analyser and evaluated in accordance with respective DSTU standard. Freezing resistance of sperm was estimated as a percentage ratio of sperm doses rejected after cryopreservation to their total amount. The absolute sperm efficiency of stud bulls was evaluated by the method of the experts of the Institute of Animal Breeding and Genetics nd. a. M.V. Zubets of National Academy of Agrarian Science. It was found that that stud bulls of LLC “Ukrainian Genetic Company” are characterized by sufficient fertility. They have produced, on average, 156 ejaculates within a year, which included 106 high-quality ejaculates (67.9%). Characteristics of sperm efficiency of experimental stud bulls are rather variable (Сv from 23.1-79.7%), and vary within the following limits: the number of high-quality ejaculates throughout a year – 32-173 pcs., native sperm obtained – 201-1016 ml, percent of rejected sperm – 3.1-76.1, doses of sperm obtained – 5755-61920 pcs. It was revealed that probable difference in sperm production indicators of sperm providers is associated with their individual characteristics. Volume of ejaculate ranges from 3.77 to 7.30 ml (d=3.57; td=16.6; P<0.001), sperm motility ranges from 7.2 to 8.3 points (d=1.1; td=11.1; P<0.001), sperm concentration in ejaculate ranges from 1.51 to 3.52 bln/ml (d=2.01; td=24; P<0.001), freezing resistance of sperm ranges from 59.6 to 99.6%. Holstein stud bulls of LTD “Ukrainian Genetic Company” have rather high sperm productivity index that ranges from 5.19 to 15.29 bln of motile spermatozoids in ejaculate. A strong positive correlation was found between motility and sperm concentration (r = +0.825; P<0.001), motility and the quantity of sperm doses obtained per year (r = +0.766; P<0.001), concentration and quantity of sperm doses obtained per year (r = +0.595; P<0.001)

Use of Acetylcysteine in the Treatment of Patients with Chronic Prostatitis/Chronic Pelvic Pain Syndrome

Antioxidants are recommended for the treatment of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). The efficacy and safety of quercetin in the treatment of patients with CP/CPPS have been studied most. To increase the effectiveness of treatment, we propose to use acetylcysteine, which, along with antioxidant properties, has secretolytic properties.The objective: to provide a comparative assessment of the efficacy and safety of quercetin and acetylcysteine in the treatment of patients with CP/CPPS.Materials and methods. The study involved 60 patients with CP/CPPS aged 18–45 years. Patients were randomized into two comparison groups. Patients in group 1 (n=30) were prescribed quercetin1 g per day for 1 month. Patients in group 2 (n=30) received acetylcyteine orally at a dose of 600 mg per day for 1 month. The condition of patients was assessed according to the NIH-CPSI scale, spermogram parameters and dynamics interlekin-10 (IL-10) and tumor necrosis factor α (TNF-α) in ejaculate. The efficiency criterion was considered to be a 25 % decrease in the NIH-CPSI.Results. As a result of the treatment of patients with CP/CPPS with the use of antioxidants in both groups symptoms of the prostatitis were reduced: NIH-CPSI total (group 1 – 22,5±0,5 vs. 15,0±0,8; group 2 – 22,7±0,5 vs. 15,1±1,1); pain (group 1 – 11,5±0,3 vs. 7,1±0,4; group 2 – 10,0±0,5 vs. 6,6±0,6); urinary symptoms (group 1 – 3,4±0,3 vs. 2,1±0,2; group 2 – 4,8±0,4 vs. 2,2±0,4), quality of life (group 1 – 7,7±0,4 vs. 5,8±0,3; group 2 – 7,8±0,3 vs. 6,3±0,4). The fertility was improved significantly (p<0,05): increased sperm concentration, progressive sperm motility, decreased number of pathological sperm, leukocyte TNF-α concentration and increased IL-10 concentration in ejaculate. In group 2 only ejaculate viscosity decreased significantly (p<0,05), improved sperm motility, decreased white blood cell count, decreased TNF-α concentration in ejaculate were significantly greater than in group 1. The clinical efficacy of acetylcysteine and quercetin in the treatment of patients with CP/CPPS was 63,3 % and 60,0 % respectively (subject to the efficacy criterion of reducing the NIH-CPSI score by 25 %).Conclusion. The use of acetylcysteine can be useful for patients with CP/CPPS.

Dependence of Sperm Morphology and Ejaculate Characteristics on Sperm Concentration in the Ejaculates of Hypor Boars.

IntroductionDetermination of sperm concentration and morphology in ejaculate is essential in evaluating fertility. Sperm shape and dimensions may depend on their concentration in ejaculate. The aim of the study was to evaluate the dependence of Hypor boar morphological and morphometric sperm characteristics on concentration in ejaculate.Material and MethodsThe study was conducted on 120 ejaculates from 12 Hypor boars. Depending on sperm concentration, they were put into three groups: low, medium, and high. Ejaculate sperm concentration was determined with the photometric method. Slides were prepared from semen samples which were evaluated for the morphology of spermatozoa. The preparations for morphological analyses were by the eosin-gentian staining method.ResultsAs the sperm concentration in the ejaculate increased, the length, perimeter, and area of the sperm heads also did. In the ejaculates with higher sperm concentration the sperm heads were rounder. The ratios of head dimensions to tail length or total length were the highest in the sperm from ejaculates with the highest concentrations. The highest percentage of morphologically abnormal sperm was noted in ejaculates with low concentrations.ConclusionThe dimensions and shape of sperm depend on the sperm concentration in the ejaculate. In ejaculates with the highest concentrations, the sperm have larger dimensions. Sperm concentration affects the frequency of morphological anomalies in the spermatozoa.

Relationship of arctic fox (Alopex lagopus L.) sperm morphology with age of males, sperm concentration, ejaculate volume and acrosin activity

Abstract The aim of the study was to determine the relationship of sperm morphology with age of males, ejaculate concentration and volume, as well as with acrosin activity determined in sperm acrosome extracts. The study used manually collected ejaculates from 9 male arctic foxes, including 6 young males aged one year and 3 older males (between 3 and 5 years of age). All of the 39 ejaculates used in the study were classified as normal based on motility exceeding 70%. The ejaculates collected from the foxes were evaluated for volume, sperm concentration and frequency of morphological changes including primary and secondary defects. The spermiograms of the male arctic foxes were classified according to a six-grade subjective scale. In addition, acrosin activity was determined in the sperm acrosome extracts. The data were analysed using the criteria of male age, sperm concentration, ejaculate volume, and acrosin activity. The morphology of arctic fox spermatozoa was dependent on the age of the male. A greater number of morphologically altered spermatozoa tended to occur in the ejaculates of young foxes, which were in their first breeding season. In addition, statistical analysis revealed positive relationships between the frequency of morphological changes in sperm and their ejaculate concentration. In contrast, there were no significant correlations between the percentages of morphologically changed spermatozoa and the ejaculate volume and the content of acrosin, which is an indicator of acrosomal integrity. Semen quality is dependent on the number of sperm in ejaculate with morphological defects which prevent oocyte fertilization. Therefore, morphological assessment of semen, which covers both the number and type of morphological changes, is highly useful when selecting appropriate males for reproduction.

Novo protocolo utilizando ocitocina para induzir a ejaculação em garanhão penectomizado

Background: Several reproductive diseases can prevent ejaculation by the traditional method of collection. Neoplasias as squamous cell carcinoma is the most common tumor of the external genitalia of horses and its lesions usually prevent copulation. The pharmacological induction of ejaculation is an important alternative technique to obtain and preserve the genetic material of stallions incapable of ejaculating by traditional methods of semen collection. However, the protocols currently used have shown questionable results and new protocols are needed in order to increase the success rates. The aim of this study is to report the success of a new protocol in inducing ejaculation when oral imipramine and intravenous oxytocin and detomidine were administrated in a Crioulo stallion.Case: A 9-year-old Crioulo stallion was admitted at the Veterinary Hospital of the São Paulo State University, FMVZUNESP, Botucatu, Brazil, with a history of a mass located on the glans and body of the penis. The histopathological exam confirmed the diagnostic of Squamous cell carcinoma and penectomy was performed. After 10 days of surgery the stallion was submitted to 5 different protocols with 3 days interval between the follow protocols: Imipramine+Xylazine; Imipramine+ Xylazine+Oxytocin; Imipramine+Detomidine and Imipramine+Detomidine+Oxytocin.Discussion: The traditional protocol of pharmacologically-induced ejaculation with imipramine hydrochloride (3 mg/kg/v.o) and xylazine hydrochloride (0.66 mg/kg/iv) was not successful even when oxytocin (20 UI/iv) was added to this protocol. Administration of imipramine hydrochloride (3 mg/kg/v.o) two hours prior to administration of detomidine hydrochloride (0.02 mg/kg/i.v) also did not result in ejaculation. However, administration of imipramine hydrochloride (3 mg/kg/v.o) 2 h prior to administration of detomidine hydrochloride (0.02 mg/kg/i.v) associated with oxytocin (20 U.I/i.v) resulted in ejaculation. The stallion was submitted to three seminal collections with a three-day interval between administration of the protocol and ejaculated in all the attempts after approximately 5 min of detomidine and oxytocin injection, presenting mean values of 50 mL of total volume (TV) and concentration of 80x106 spermatozoa/mL. The VT was higher and concentration was lower when compared to ejaculates obtained by pharmacological induction in previous studies, probably due to daily stimulation with estrus mare to induce penile exposure in order to allow antisepsis of the surgical wound. Thus, it is believed that the large amount of total volume of this stallion is due to the high production of gel by the accessory sex glands, with consequent reduction of ejaculate concentration. The sperm kinetics were evaluated by the computerized method CASA (HTMA-IVOS-12) with total motility (MT) of 84% and progressive (MP) of 38%, with 70% of rapid spermatozoa (RAP), being considered normal to the equine specie and similar to those observed by other authors in pharmacolocally-induced ejaculates. Post-thaw sperm kinetics presented 42% of MT, 21% of MP and 28% of RAP probably due to an intrinsic sensitivity of the stallion to the freezing process. Thus, this report concludes that the protocol associating imipramine, detomidine and oxytocin was efficient in the pharmacological induction of ejaculation, presenting normal and characteristic sperm parameters of the specie. Fresh and refrigerated semen presented good parameters for use in conventional artificial inseminations while frozen semen is indicated for deep horn inseminations or for use in intracytoplasmic sperm injection (ICSI) programs.

Effect of human menopausal gonadotropin on testicular morphometry, gonadal and extragonadal sperm reserves of rabbit bucks

<p>Testicular morphometry, gonadal and extragonadal sperm reserves of rabbit bucks treated with Human Menopausal Gonadotrophin (Menogon®) were studied. Twenty-four crossbred rabbit bucks weighing 1.3-1.6 kg at 15-17 wk were randomised into 4 treatment doses of 0 (control), 7.5, 15.0, and 22.5 I.U. in a completely randomised design with 6 bucks per group. A vial of Menogon® was reconstituted in 1 mL of physiological saline solution and administered intramuscularly at 72 h intervals for 56 d prior to commencement of sample collection. Parameters evaluated were ejaculate concentration, testicular morphometry, gonadal and extragonadal sperm concentrations. Results for ejaculate concentration (86, 110, 186, and 135×106/mm3) revealed a significantly (P<0.05) higher difference in the 15.0 I.U. group. Gonadal and extragonadal sperm reserve indices were not significantly (P>0.05) affected by the treatment. Testicular morphometry results showed that paired testis volume (cm3), testis circumference (cm), ductus deferens length (cm) and epididymal length (cm) were significantly (P<0.05) different, with a progressive increase that corresponds to increased Menogon® administration. High and positive correlations (P<0.01) were observed between testis weight and gonadal sperm reserves (r=0.99) and body weight and gonadal sperm reserves (r=0.99). Similarly, correlations between epididymal weight and epididymal sperm reserves (r=0.85), testis weight and ejaculate concentration (r=0.97), body weight and ejaculate concentration (r=0.96), body weight and testis weight (r=0.97), testis volume and ejaculate concentration (r=0.97), testis volume and gonadal sperm reserve (r=0.91), testis volume and testis weight (r=0.96), and testis volume and body weight (r=0.90) were positive and significant (P<0.05). Taking all the reported results into consideration, controlling the amount of Menogon® administration at 15.0 I.U. for rabbit bucks could be a promising approach to regulating testosterone synthesis and secretion, thereby affording a potential method of enhancing fertility.</p>

CRYOPRESERVATION OF RED BROCKET DEER SEMEN (MAZAMA AMERICANA): COMPARISON BETWEEN THREE EXTENDERS

In recent years, genome banks have grown as a way of maintaining the genetic variability of populations. However, the quality of gamete cryopreservation will determine their efficiency. This study aimed to evaluate prefreeze and postthaw sperm motility, vigor, membrane integrity, and morphology of semen of red brocket deer (Mazama americana) using three extenders: E1, Tris-Yolk; E2, Tes-Tris-Yolk; and E3, Tes-Tris-Yolk-Equex. Six bucks were used, and three collections per buck were performed at 90-day intervals. Before freezing, semen volume, ejaculate concentration, motility, vigor, membrane integrity, and sperm morphology were evaluated. To compare the effect exerted by the extenders after sample thawing, further analyses of sperm motility, vigor, membrane integrity, and morphology were performed. Mean ejaculate volume and sperm concentration were 365.33 +/- 120.5 microL and 2,675.73 +/- 810.4 sperm/mL, respectively. Prefreeze motility for the extenders showed no significant differences (approximately 60%). Postthaw motility (E1 = 16.33 +/- 5.5, E2 = 5.44 +/- 5.2, E3 = 24.66 +/- 10.0) was significantly different between E2 and E3, whereas postthaw vigor (E1 =2.66 +/- 0.8, E2= 1.89 +/- 1.2, E3 = 3.83 +/- 0.4) was greater for E3 (P < or = 0.05). Analysis of postthaw membrane integrity revealed no significant differences between the extenders regarding counts of cells presenting intact membranes; however, E3 promoted the lowest number of cells with damaged membranes and higher cell counts for partially damaged membranes (P < or = 0.05). Analysis of sperm morphology revealed an increase in severe abnormalities when using E2 and E3 (P < 0.05). However, observation verified that counts of altered cells were lower using E3 than E2, suggesting a protective effect of Equex. These findings indicate that E3 promoted better semen quality postthaw. However, the performance of this extender in protecting sperm cells of M. americana during freezing was lower than that verified for other species. Thus, further studies are needed for characterization of red brocket deer semen and the optimization of extender for sperm cryopreservation.

Comparação de diferentes diluentes na mensuração da concentração espermática de suínos em espectrofotômetro

To obtain success in an artificial insemination (AI) program, it is essential that the AI centrals produce high quality insemination doses (ID); one important factor is to use the correct sperm concentration by ID. To determine sperm concentration, spectrophotometry could be used. This technique consists in the reading of sperm concentration through the measurement of light transmittance percent in the ejaculate sample. Usually, when executing this technique, cancerigenous solutions, as formalin, or neurotoxic solutions, as glutaraldehyde, are used. This work aimed to compare different diluent solutions to measure swine sperm concentration on the spectrophotometer. Fifty three swine male breeders from different strains and with proven fertility were used. Ejaculates were collected by the gloved hand method. A spectrophotometer (QUIMIS Q-798DMR), previously calibrated with the semen from the animals of the own farm, was used. In the cuvettes, 0.03 mL of the ejaculate sample was added to 2.97 mL of saline phormol (T1) or physiologic solution (T2). Data was analyzed by ANOVA using Statistix 2003 software. There was no difference between treatments (P &gt; 0,05). Mean concentration of 5.52x106 ± 75.0 and 5.50x106 ± 68.0 sperm/mL, corresponding to 68.2 ± 2.5 and 67.1 ± 2.5 % transmittance were observed for T1 and T2, respectively. Replacing saline formalin with physiologic solution does not bring losses in the results and keeps the laboratory formalin-free. Therefore, using physiologic solution to read ejaculate concentration on the spectrophotometer is more indicated in swine AI centrals.

280 BOVINE SPERM DNA FRAGMENTATION RATES AND EXTENDER pH: A DYNAMIC EXPERIMENTAL APPROACH

Flow cytometry technology for the sorting of X- and Y-chromosome bearing sperm is currently utilised in research and for commercial applications. During sample preparation before the sex-sorting process, sperm go through different pH treatments that may affect their quality. The hypothesis to test in this study is that sperm DNA damage could occur due to differences in the pH of sperm extenders. Bull semen doses from 15 dairy and 15 beef bulls (2 ejaculates per individual), ranging in age between 13 and 96 months, were randomly selected from a bull stud in Texas (Sexing Technologies, Navasota, TX, USA). Each semen sample was divided into 4 separate aliquots. One neat semen sample was kept as a control and the other 3 aliquots were treated with 16 μL of 8.1 mM Hoechst 33342 (Molecular Probes, Eugene, OR, USA). A calculated amount of modified Tyrode’s albumin lactate pyruvate (Clear TALP), pH 7.4, was added based on neat ejaculate concentration. For the separation of live and dead sperm during the sex-sorting process, 2 mL of red TALP (Red Food Dye FD&amp;C #40; Sensient Technologies Corp., Milwaukee, WI, USA) at 3 different pH treatment levels (5.5, 6.4, and 7.4) were added to the sperm samples. The dynamics of sperm DNA fragmentation were assessed immediately following the addition of red TALP treatment and after 24 h of incubation at 34°C. Sperm DNA fragmentation was measured using the commercial variant of the sperm chromatin dispersion test, the bull Sperm-Halomax® kit (Halotech DNA, Madrid, Spain), and counting 300 sperm cells under fluorescence microscopy. Analysis of variance was used to determine if there were statistical differences (α = 0.05) among mean values of the groups (SPSS v.17.0 for Windows, SPSS Inc., Chicago, IL, USA). Bonferroni post hoc tests were utilised to determine the pairwise directional differences between groups. Differences in DNA fragmentation were not observed at 0 h (P &gt; 0.05); however, at 24 h this value was significantly higher (P &lt; 0.05) in semen samples treated with red TALP, pH 6.4, v. samples treated with red TALP, pH 7.4, (44.0 ± 24.9% v. 34.8 ± 24.9%), and even higher when using red TALP pH 5.5 (50.8 ± 23.8%). Further experiments should be designed to demonstrate that the DNA molecule could be affected by pH fluctuations, which may be important in refining the sex-sorting process. Notably, the undiluted neat semen controls had greater levels of DNA fragmentation overall (57.5 ± 29.4%), suggesting a negative effect of high sperm concentrations and seminal plasma. The authors thank Mike Evans, Eddy Valenzuela, David Del Olmo, Jamie Jeffers, and the staff at Sexing Technologies for their technical assistance. This research was funded by Sexing Technologies and XY Inc.

EXPERIENCE OF THE THERAPY OF HYPOANDROGEN MALE INFERTILITY WITH TAMOXIFEN — AEVIT COMBINATION

Studies into peculiar changes of spermatogram parameters, fructose content in ejuaculate and hormonal levels of hypophysic-gonadal system using the therapy of idiopathic pathospermias in hypoandrogenized males treated with Tamoxifen-Aevit combination have been performed. The applied therapy led to a profound improvement of spermatogram parameters, testosterone secretion stimulation, increased fructose concentration in ejaculate. Tamoxifen combined with Aevit had a more substantial influence upon normalization of all spermatogram parameters of infertile males when prior to their treatment the testosterone-luteinizing hormone ratio exceeded 2,0.

The effectiveness of modified sperm washes in severely oligoasthenozoospermic men infected with human immunodeficiency and hepatitis C viruses

Men who are seropositive for human immunodeficiency virus (HIV) or hepatitis C virus now can father children safely, thanks to sperm washing and nested polymerase chain-reaction techniques, followed by intracytoplasmic sperm injection. However, in a small percentage of such patients, it is impossible to recover spermatozoa after said procedures because of their highly impaired spermatogenesis. We have established that less rigorous methods, such as repeated centrifugation, yield nested polymerase chain-reaction HIV- and hepatitis C virus-negative specimens, even in sperm samples from men with severe oligoasthenozoospermia.

Monthly variations in ovine seminal plasma proteins analyzed by two-dimensional polyacrylamide gel electrophoresis

This study was conducted to evaluate monthly changes in the ram seminal plasma protein profile using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with a polyacrylamide linear gradient gel. Likewise, comparative analyses of the protein composition of ovine seminal plasma (SP) from ejaculates obtained along the year, and its relationship with sperm motility, viability and concentration of ejaculate were carried out. Western-blot analysis was performed to specifically detect P14, a ram SP protein postulated to be involved in sperm capacitation and gamete interaction [Barrios B, Fernández-Juan M, Muiño-Blanco T, Cebrián-Pérez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins which protect ram spermatozoa against cold-shock. J Androl 2005;26:539–49], and its variations along the year have also been established. The experiment was carried out from May 2003 to April 2004, with nine Rasa Aragonesa rams. Ejaculates obtained every 2 days were pooled and used for each assay, to avoid individual differences, and three two-dimensional SDS-PAGE gels were run for each month. The high resolution of the gradient gel allowed the image analysis software to detect around 252 protein spots, with pIs ranging from 4.2 to 7.6, and molecular weight (Mr) from 12.5 to 83.9kDa. Four protein spots (1, 2, 3 and 4) of low Mr (15.1, 15.7, 15.9 and 21.0kDa) and acidic pI (5.9, 5.3, 5.7 and 6.6), respectively, had the highest relative intensity in the SP map (11.2, 9.3, 4.7 and 7.7%, respectively). Spot 3 was more abundant (P<0.05) from May to December, and negatively correlated (P<0.05, r=−0.34) with sperm viability and concentration (P<0.05, r=0.36). Another 12 protein spots also had significant quantitative differences (P<0.05) along the year, and 17 protein spots, which correlated with some seminal quality parameter, did not show quantitative monthly changes. Western-blot analysis indicated that spots 1 and 2 reacted with the anti-P14 antibody, raised against the P14 band (approximate Mr 14kDa) of ram SP. This indicates that spots 1 and 2 are similar to RSP15 [Bergeron A, Villemure M, Lazure C, Manjunath P. Isolation and characterization of the major proteins of ram seminal plasma. Mol Reprod Dev 2005;71:461–70], bovine PDC-109 [Esch FS, Ling NC, Bohlen P, Ying S, Guillemin R. Primary structure of PDC-109, a major protein constituent of bovine seminal plasma. Biochem Biophys Res Commun 1983;113:861–7] (also called BSP A1/A2 [Manjunath P, Sairam MR. Purification and biochemical characterization of three major acidic proteins (BSP-A1, BSP-A2 and BSP-A3) from bovine seminal plasma. Biochem J 1987;241:685–92]) and goat GSP-14/15kDa [Villemure M, Lazure C, Manjunath P. Isolation and characterization of gelatine-binding proteins from goat seminal plasma. Reprod Biol Endocrinol 2003;1:39], based on our previous results on the P14 amino acid sequence [Barrios B, Fernández-Juan M, Muiño-Blanco T, Cebrián-Pérez JA. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins which protect ram spermatozoa against cold-shock. J Androl 2005;26:539–49].

Concentrations of cefpodoxime in plasma, ejaculate and in prostatic fluid and adenoma tissue

Twenty-four healthy volunteers and 24 patients undergoing transurethral resection of the prostate received an oral dose of 200 mg of cefpodoxime as proxetil ester in a fasting state. At the same time 3.235 g of iohexol, a renal contrast medium, was injected intravenously to indicate possible urinary contamination of the prostatic fluid. The subjects were divided into three groups each. After 3, 6 and 12 h the cefpodoxime concentrations were measured in plasma, urine, prostatic fluid and ejaculate in volunteers and in plasma, prostatic fluid and prostatic adenoma tissue in patients by a bioassay as well as by an HPLC method. In general, the concentrations measured by bioassay were higher than those by HPLC. The median plasma concentrations (bioassay) in volunteers (patients) after 3, 6 and 12 h were 2.28 (2.34) mg/l, 0.95 (1.17) mg/l and 0.12 (0.28) mg/l, respectively. The median ejaculate concentrations after 6 and 12 h were 0.95 mg/l and 0.19 mg/l, respectively. Only in three volunteers and in one patient prostatic fluid concentration without urinary contamination could be measured after 3 h with a median fluid to plasma ratio of 0.10. The prostatic adenoma tissue concentrations (bioassay) after 3 and 6 h were 0.50 mg/kg and 0.24 mg/kg with tissue to plasma ratios of 0.30 and 0.26, respectively. After 3 h about half of the volunteers and after 12 h about half of the patients showed no detectable concentration in ejaculate (volunteers) and prostatic tissue (patients), respectively. It was concluded that the cefpodoxime should be administered 3 to 6 h prior to surgery if used for perioperative prophylaxis.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic and Environmental Components of Semen Production Traits of Artificial Insemination Holstein Bulls

A multiple trait procedure allowed joint estimation of model effects and variance and covariance components of semen production of Holstein bulls in artificial insemination. Procedural advantages included rapid iteration and positive definite estimates of covariance matrix. Data consisted of 149,339 semen production records for total sperm, volume, and concentration of ejaculate on 2,351 bulls collected in three bull studs from July 1969 through November 1982. Models defined to describe the biology of semen production included effects for ejaculates, age of bull, ambient temperature, week day by bull barn, scheduling, month by year, colorimeter by calibration period, and bulls. Environmental trends but no genetic trends in semen production were detected. Repeatabilities of measures of total sperm, volume, and concentration of all ejaculates were .26, .23, and .37 and for first ejaculates were .31, .23, and .42 over all studs. Heritabilities were .03, .18, and .10 for all ejaculates and .05, .16, and .16 for first ejaculates. Repeatabilities of first ejaculate measures for bulls collected up to 15 mo and again from 60 mo of age were .21, .27, and .19. Genetic correlations between first ejaculate measures on young bulls and milk and milk fat production in daughters ranged from −.06 to .05.