Effect of whey protein supplementation on sperm quality and fertility in male mice.

Protein supplements are a billion-dollar industry and the intake of these supplements is increasing, especially among young men. However, little is known about whether consumption of these products affects the reproductive health. The aim of this study was to assess the effect of whey protein supplementation on the sperm quality and reproductive health of male mice. A total of 48 male NMRI mice were fed with either plain tap water or a high dose of whey protein (Whey100, BodyLab) supplemented in the drinking water for 3 months. Mice was individually housed with two female mice for five days and reproductive parameters were assessed. DNA fragmentation index (DFI) was assessed at 0 h and 4 h of in vitro incubation using a sperm DNA integrity test (SDI®-test). No significant differences were detected between the groups in the epididymal sperm count, sperm motility, DFI, oxidation-reduction potential (ORP), serum testosterone, body and seminal vesicles weights, relative testis and epididymal weights, testicular morphology, number of impregnated females, or litter size. No correlation was found between ORP and DFI. These results suggest that the highest recommended human dose of whey protein supplementation do not significantly impair the sperm quality and fertility in male mice.

45,X/46,XY Mosaicism and Normozoospermia in a Patient with Male Phenotype.

The phenotypic spectrum of 45,X/46,XY mosaic males varies greatly. Previous reports have only described cases with either oligozoospermia, growth retardation, or elevated gonadotropins. However, the present case presented with normozoospermia, and normal height, sperm DNA fragmentation index (DFI), and gonadotropins. The male and his spouse were referred to The Fertility Clinic, Skive Regional Hospital, due to 2 years of infertility. After failure of several attempts of assisted reproductive treatment (ART), the male underwent genetic analysis. Conventional karyotyping in peripheral lymphocytes yielded a low-grade 45,X/46,XY mosaicism, confirmed by fluorescence in situ hybridization (FISH) showing 6% 45,X cells. A FISH test performed on interphase nuclei from buccal mucosal cells yielded one cell with only one X-signal (0.6%), explaining the normal phenotype of the patient, but not the infertility. FISH test for sperm aneuploidy showed normal range parameters, except for a 10-fold elevated gonosomal nullisomy rate (2.1%). Hence, germinal mosaicism may be an explanation of the infertility of the case. Increased sex nullisomy levels may reflect an aberrant testicular environment compromising fertility even though sperm euploidy rates and other sperm parameters do not preclude a successful treatment with ART. Based on these results, the couple decided to use donor semen for their subsequent intrauterine insemination treatment and obtained a successful pregnancy.

Pulmonary exposure to carbonaceous nanomaterials and sperm quality.

BACKGROUND: Semen quality parameters are potentially affected by nanomaterials in several ways: Inhaled nanosized particles are potent inducers of pulmonary inflammation, leading to the release of inflammatory mediators. Small amounts of particles may translocate from the lungs into the lung capillaries, enter the systemic circulation and ultimately reach the testes. Both the inflammatory response and the particles may induce oxidative stress which can directly affect spermatogenesis. Furthermore, spermatogenesis may be indirectly affected by changes in the hormonal milieu as systemic inflammation is a potential modulator of endocrine function. The aim of this study was to investigate the effects of pulmonary exposure to carbonaceous nanomaterials on sperm quality parameters in an experimental mouse model. METHODS: Effects on sperm quality after pulmonary inflammation induced by carbonaceous nanomaterials were investigated by intratracheally instilling sexually mature male NMRI mice with four different carbonaceous nanomaterials dispersed in nanopure water: graphene oxide (18 µg/mouse/i.t.), Flammruss 101, Printex 90 and SRM1650b (0.1 mg/mouse/i.t. each) weekly for seven consecutive weeks. Pulmonary inflammation was determined by differential cell count in bronchoalveolar lavage fluid. Epididymal sperm concentration and motility were measured by computer-assisted sperm analysis. Epididymal sperm viability and morphological abnormalities were assessed manually using Hoechst 33,342/PI flourescent and Spermac staining, respectively. Epididymal sperm were assessed with regard to sperm DNA integrity (damage). Daily sperm production was measured in the testis, and testosterone levels were measured in blood plasma by ELISA. RESULTS: Neutrophil numbers in the bronchoalveolar fluid showed sustained inflammatory response in the nanoparticle-exposed groups one week after the last instillation. No significant changes in epididymal sperm parameters, daily sperm production or plasma testosterone levels were found. CONCLUSION: Despite the sustained pulmonary inflammatory response, an eight week exposure to graphene oxide, Flammruss 101, Printex 90 and the diesel particle SRM1650b in the present study did not appear to affect semen parameters, daily sperm production or testosterone concentration in male NMRI mice.

Dynamics of the induced acrosome reaction in boar sperm evaluated by flow cytometry.

The present study investigated the dynamics of the in vitro induced acrosome reaction (AR) in boar sperm in response to medium composition, incubation time and ionophore concentration. The AR is a prerequisite for normal sperm fertilizing capability and can be studied in vitro following induction by various agents. The ability of a sperm population to undergo the AR in vivo is expected to influence male fertilizing potential, and attempts to relate the in vitro induced AR to fertility has been reported. However, to relate the induced AR to fertility one should be aware of the dynamics of the in vitro induced AR. A detailed description of the dynamics of sperm viability and acrosomal status of boar sperm following in vitro induction of the AR has to our knowledge not previously been conducted. In the present study, a triple color flow cytometric detection technique was used, which gave simultaneous information on sperm viability and acrosomal status. The ionophore induced AR was dependent on extracellular Ca(2+), but could be easily induced in boar sperm without capacitation. Capacitation-associated plasma membrane phospholipid scrambling was assessed and a medium specific ability to induce these membrane changes was observed. Both sperm viability and the induced AR were significantly affected by sperm capacitation, incubation time and ionophore concentration. The results lead to suggestions for an optimized AR induction protocol that takes both sperm viability and the effectiveness of AR induction into consideration.

The sperm chromatin structure assay as a diagnostic tool in the human fertility clinic.

BACKGROUND: Sperm DNA integrity has been shown to be necessary for achieving and sustaining embryo development. The objective was to evaluate the sperm chromatin structure assay (SCSA) as a diagnostic tool in clinical practice for intrauterine insemination (IUI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatments. METHODS: A total of 385 semen samples from 234 couples were frozen for SCSA, and smears were prepared for morphology: 48 IUI, 139 IVF and 47 ICSI. The main SCSA variables were DNA fragmentation index (DFI), standard deviation of DFI (SD-DFI) and high DNA stainability (HDS), and the reproductive outcomes were biochemical pregnancy (BP), clinical pregnancy (CP) and implantation ratio (IR). RESULTS: The results showed no significant difference in the fertility variables BP, CP and IR when <27% DFI was used between the IVF and ICSI groups. A low number of patients received IUI with low success rate, and statistical analysis was therefore not performed. Ongoing pregnancy was achieved for both IVF and ICSI couples with DFI levels >27%, and six couples in ICSI treatment achieved CP full-term. DFI >27% had a high prognostic power for predicting no CP for IVF patients, with a specificity of 97%. Couples diagnosed with male infertility had a significantly higher level of DFI compared to couples with idiopathic fertility. Sperm head morphology showed low but significant correlations with the SCSA variables. CONCLUSION: SCSA is a useful tool in andrological diagnosis and contributes with a prognosis for the fertility outcome of conventional IVF. Although full-term pregnancy can be achieved with assisted reproductive techniques with a DFI >27%, the probability of a successful pregnancy may be reduced.

Variability and laboratory factors affecting the sperm chromatin structure assay in human semen.

During the past decade, the sperm chromatin structure assay (SCSA) has become an important tool for assessing semen quality in the human andrology laboratory. The SCSA uses the metachromatic properties of the fluorescent dye acridine orange (AO) in combination with flow cytometry to determine the sperm DNA susceptibility to denaturation in situ. The objective of this study was to evaluate laboratory factors affecting the SCSA and the variation between replicates. Semen ejaculates from 3 healthy volunteers were analyzed using the SCSA protocol as described by Evenson and Jost (2000), determining the X-mean, Y-mean, DNA fragmentation index (DFI), standard deviation of DFI (SD-DFI), and high DNA stainability (HDS). In experiment 1, the effects of thawing time, time of day, day, laboratory technician, donor, and incubation period before analysis were investigated. In experiment 2, the effects of sheath fluid, AO equilibration buffer, day, laboratory technician, donor, and incubation period before analysis were investigated. A significant difference was found between the 3 donors with respect to the X-mean, Y-mean, DFI, SD-DFI, and HDS. It was shown that incubation of the semen samples on ice postthaw had a significant effect on the X-mean, Y-mean, DFI, and SD-DFI. The laboratory technician conducting the analysis accounted for up to 15.4% for the variation of the SCSA measurements. The time of day affected the variation for the Y-mean (23.5% of the total variation of the Y-mean), and the day affected the variation for the X-mean (82.8% of the total variation of the X-mean). Incubation on ice for 5 to 25 minutes postthaw had a significant effect on the DFI and SD-DFI in both experiments. This study shows that several protocol steps in the SCSA affect the results obtained from the assay. Precise protocol description and standardization of the SCSA are therefore essential to achieve high agreement within and between different laboratories.

Increasing storage time of extended boar semen reduces sperm DNA integrity.

There is an extensive use of artificial insemination (AI) in the pig industry. Extended liquid boar semen may be used for insemination for up to 5 days after collection. The objective of this study was to determine the changes in sperm quality, when boar semen was extended and stored at 18 degrees C for up to 72 h post-collection. The study included three ejaculates from five boars, for each of the four breeds: Duroc, Hampshire, Landrace and Danish Large White (n=60 ejaculates). The sperm chromatin structure assay (SCSA) showed an increase in DNA fragmentation index (DFI) after 72 h of incubation (P<0.001), with no differences between breeds (P=0.07). For two Hampshire boars, all ejaculates had a large increase in DFI after 24 h of incubation. The standard deviation of DFI (SD-DFI) differed between breeds, with the SD-DFI for Hampshire being significantly greater than for the other breeds. The SD-DFI did not change during the 72 h of storage. Sperm viability was determined using SYBR-14 and propidium iodide in combination with flow cytometry. The sperm viability did not differ between breeds (P=0.21), but a difference in viability during storage (P<0.001) was detected. In conclusion, the SCSA cytogram patterns were consistent for different ejaculates within boars and storage of extended boar semen at 18 degrees C for 72 h significantly decreased the integrity of sperm DNA.

DNA integrity in sexed bull sperm assessed by neutral Comet assay and sperm chromatin structure assay.

During the production of sex-sorted spermatozoa from bull semen, the cells are exposed to a number of potential hazards including: dilution, centrifugation, incubation, exposure to DNA stains and laser light. These factors may affect the survival capacity and fertilization potential of the sperm. The objective of this study was to determine whether sex-sorted bull spermatozoa have more DNA damage than sperm from conventional processed bull semen. Two methods were used to determine DNA integrity: the neutral Comet assay (NCA) and the sperm chromatin structure assay (SCSA). The NCA showed that the conventional samples had a higher tail moment (TM) (P < 0.017) than the sorted samples and that there was no difference between the samples in tail length (TL) (P = 0.36). The SCSA showed that the DNA fragmentation index (DFI) was higher for conventional than the sorted samples (P = 0.011), but the standard deviation of DFI (SD-DFI) was higher for the sorted samples (P < 0.001). We conclude that the NCA and SCSA can be used in assessing DNA integrity in bovine sperm and that cell sorting by flow cytometry improves the integrity of the sperm cell population. Additionally the results from the SCSA indicated that the sex-sorted sperm had less homogenous sperm chromatin. In the future assessment of sperm DNA integrity may be used to select bulls for sperm sex sorting and optimizing sperm sex sorting procedures.

Relationship between sperm viability as determined by flow cytometry and nonreturn rate of dairy bulls.

A newly developed flow cytometric method for determination of sperm concentration and viability was tested in an insemination trial with cryopreserved bull sperm to establish the relationship between sperm viability and nonreturn rates. Semen for experimental inseminations was produced from 157 young sires (114 Holstein and 43 Jersey), each contributing 4 experimental semen collections. Straws containing approximately 15 x 10(6) motile sperm before freezing were used in 118,680 experimental inseminations performed by 254 artificial insemination technicians in 6352 Danish herds. Statistical analysis based on 44,946 experimental first inseminations showed that the major part (95.4%) of variation in the 56-day nonreturn rate (NRR56) was residual. Only 0.38% of the total variation in NRR56 was due to bulls and differences between ejaculate within bull. However, bulls were preselected, and a relatively high insemination dose was used. Correlations between sperm viability as assessed by flow cytometry and NRR56 was slightly lower than observed for microscopic assessment of sperm motility. However, flow cytometry makes it possible to achieve an objective and precise determination of sperm viability. It was therefore possible to calculate the effect on NRR56 provided selection of semen is based on the flow cytometric method. Three freezing extenders were used in this experiment, but a significant difference in NRR56 was not observed. Flow cytometric results for 1 extender (Biociphos Plus) indicated poorer sperm survival during postthaw incubation compared with Triladyl extender with whole and with clarified egg yolk.

Quality control in boar semen production by use of the FACSCount AF system.

A simple and rapid flow cytometric method has recently been developed for simultaneous determination of sperm concentration and viability in semen from domestic animals. Use of SYBR-14 trade mark in combination with propidium iodide (PI) allows estimation of the proportion of live sperm (viability). An internal standard of fluorescent microspheres (beads) makes it possible to determine the sperm concentration during the same analysis. In the first experiment, the relationship between sperm viability and litter size was investigated. The second experiment explored whether a smaller variation in the number of motile sperm per insemination dose could be obtained using the FACSCount AF flow cytometer than using a spectrophotometer. Results in the first experiment show that sperm viability is closer related to litter size than is the traditionally used motility parameter. Although the flow cytometer is precise and objective, a limited effect on litter size should be anticipated if ejaculates are selected for insemination according to the percentage of viable sperm. However, the present trial used large insemination doses (2.3 x 10(9) motile sperm/dose) which partially compensate for the differences in motility and viability between boars and ejaculates. In the second experiment it was found that variation in the number of motile sperm per insemination dose could be reduced significantly if the FACSCount AF flow cytometer rather than the Corning 254 spectrophotometer was used for determination of sperm concentration in the raw semen. It is concluded that the FACSCount AF flow cytometer is a strong tool for improvement of the quality control in artificial insemination (AI) centres.

A flow cytometric method for rapid determination of sperm concentration and viability in mammalian and avian semen.

A new flow cytometric method has been developed to rapidly determine sperm concentration and viability in semen from bulls and boars. Sperm viability was determined on the basis of staining with SYBR-14 and propidium iodide (PI), and this allowed detection of live (membrane-intact) sperm, dying (moribund) sperm, as well as dead cells. Fluorescent microspheres (beads) were used to determine sperm concentration. The use of SYBR-14 at 50 nM and PI at 12 micro M in combination with the FACSCount diluent in the counting tubes resulted in a uniform staining after 2.5-5 minutes at room temperature. Reagent staining was reproducible enough to allow subsequent semiautomated analysis of data using Attractors software. In experiment 1, this method was used to analyze semen from boars, rams, rats, rabbits, humans, and turkeys. In experiment 2, Attractors analysis was performed by the FACSCount AF flow cytometer, and sperm concentration determination with this system was compared with results obtained by a spectrophotometer and an electronic cell counter, which is routinely used by bull artificial insemination centers. When compared to microscopic counting in a hemocytometer, the FACSCount AF flow cytometer was two and four times more accurate than the spectrophotometer and the electronic cell counter, respectively. In addition, the FACSCount AF flow cytometer determined both sperm concentration (coefficient of variation 3.3%) and sperm viability (coefficient of variation 0.7%) with high precision.