Rheotaxis of sperm in fertile and infertile men.

Sperm rheotaxis refers to the ability of sperm cells to align their swimming direction with or against fluid flow. Positive rheotaxis (PR) is the tendency of sperm cells to swim against the flow. Herein, we describe sperm rheotaxis in fertile and infertile males, using a microfluidic platform and focus on rheotaxis as a potential marker of male fertility. A previously reported computer-assisted sperm analysis (CASA) plugin for Image-J was used to detect and analyze the motion of human sperm cells in microfluidic environments. The fabricated microchannels mimic the female reproductive tracts and use an image-processing program to monitor sperm swimming behavior in semen samples from fertile and infertile men. We have constructed an image-processing pipeline. The image-processing pipeline incorporated strengthens object detection and particle tracking to adapt to sperm that are out of focus while swimming on the same track. PR% was defined as the number of PR sperm cells over the number of motile sperm cells. The results showed that the percentage of PR correlates with fertility, wherein the fertile male specimens showed a higher PR% than the other groups (P < 0.05). There is no difference in progressive motility between the control group (fertile men with normal sperm analysis) and group 1 (G1; infertile men with normal sperm analysis). However, PR% was lower (P < 0.05) in the G1 group (13.5 ± 0.4%) compared to the control group (40.3 ± 3.3%) and group 2 (G2; infertile with reduced sperm motility) (15.3 ± 4.6%). Thus, PR% may be used as a novel parameter to explain infertility even in situations where basic sperm analysis following the World Health Organization (WHO) guidelines is unable to do so. We propose to use PR% as a novel parameter for sperm analysis and as a method of sperm selection in assisted reproductive technology.

High concentration of growth differentiation factor-9 (GDF-9) to the ram semen had a negative effect on the sperm positive rheotaxis.

Herein, the microfluidic device technique was used to investigate the effects of GDF-9 concentrations and exposure time on the ram sperm positive rheotaxis (PR). Semen was collected from six rams and utilized for PR, motility and sperm kinetic parameter analysis using a computer-assisted sperm analysis program with controlled flow velocity following 0, 10, 20 or 30 min of incubation at 37°C with GDF-9 (200 , 400 or 600 ng/ml; semen sample without GDF-9 was used as a control). Results revealed that there was not an interaction between effects of GDF-9 concentrations and incubation duration on PR% (p = .457) and TM% (p = .099). A simple main effects analysis showed that GDF-9 concentrations had an effect on PR% (p = .003). However, the incubation duration did not have an effect on PR% (p = .101). GDF-9 concentrations had not an effect on TM% (p = .817). By contrast, the incubation duration affected on TM% (p = .026). A higher PR% was found (p < .05) at 200 ng GDF-9 after 10 min (46.7 ± 10.3) and 20 min (45.5 ± 11.5) of incubation. After 30 min of incubation, the PR% was found lowest (p < .05) at 400 ng of GDF-9 (30.6 ± 14.1) and 600 ng of GDF-9 (32.2 ± 9.6). There was no difference (p > .05) in the sperm kinetic parameters between the four treatment groups. In conclusion, the ram sperms had the best rheotaxis properties after 10 and 20 min of incubation with 200 ng of GDF-9 and were sensitive to high concretions.

Effect of increased oxygen availability and astaxanthin supplementation on the growth, maturation and developmental competence of bovine oocytes derived from early antral follicles.

In vitro growth (IVG) culture of bovine oocyte-cumulus-granulosa complexes (OCGCs) is generally carried out for 12 or 14 days using conventional gas impermeable culture devices. The culture duration may be longer compared to follicular development in vivo. During follicular development, follicles receive oxygen from micro vessels; however, oxygen supply is limited under the culture using conventional gas impermeable devices. The purpose of this study was to investigate the effect of increasing dissolved oxygen availability using a gas permeable (GP) culture device with or without antioxidant (astaxanthin, Ax) supplementation on 8-day IVG culture systems for bovine OCGCs derived from early antral follicles. We cultured OCGCs in GP, GP supplemented with Ax (GP + Ax), and a conventional gas impermeable device (control) for 8 or 12 days. OCGC viability were significantly higher when cultured for 8 days than 12 days (p < 0.001) in all culture condition, but significant difference was not observed between groups (p > 0.05). Antrum formation rates of OCGCs were higher after 12 days than 8 days of culture in all culture condition (p < 0.001) and were significantly higher in the control than GP groups regardless of Ax supplementation (p < 0.05). Oocyte diameters were similar among day-8 GP + Ax, day-8 control and day-12 control groups (p > 0.05). Nuclear maturation rates of oocytes grown in vitro for 8 days were significantly higher in the GP + Ax group than in the control and the GP groups (p < 0.05) and similar to oocytes grown for 12 days regardless of the culture conditions (p > 0.05). The generation of reactive oxygen species in OCGCs on day 8 of IVG culture was significantly lower in the GP + Ax group than those of the GP and control groups (p < 0.05). IVG oocytes after eight days of culture developed into blastocysts, and the cleavage and blastocyst rates were similar in all treatment groups. However, in vivo-grown oocytes had significantly higher (p < 0.05) cleavage and blastocyst rates than the IVG oocytes in all groups. The present study demonstrates that increased oxygen availability using a GP culture device with Ax supplementation promotes oocyte growth and maturation competence but inhibits proliferation of granulosa cells and antrum formation compared with a conventional gas impermeable culture device, and that OCGCs can attain developmental competence after 8 days of IVG culture.

Implications of ram sperm rheotaxis analysed by microfluidics for fertility.

Rheotaxis of sperm using a microfluidic device was explored in human, mice and bull. However, the rheotaxis of ram sperm and its role in fertility are unknown. Herein, we described the sperm rheotaxis in ram using microfluidic devices and focused on rheotaxis as potential markers of in vivo fertility. Computer-assisted sperm analysis (CASA) with controlled flow velocity was used to explore the kinematic parameters of sperm, total motility and positive rheotaxis (PR). The percentage of PR was defined as the number of PR sperm cells over the number of motile sperm cells. Then, according to the percentage of PR sperm, rams were classified into two groups; sperm with ≥40% PR and <40% PR, although the two ram groups showed similar total motility and kinematic values of sperm evaluated by CASA (p > .05). Two groups of rams mated one hundred thirty ewes naturally (10 ewes/ram). In the results, the pregnancy rate was higher in ≥40% PR (94.4%) than in <40% PR (42.5%, p < .05) after natural mating. Besides, the pregnancy loss was higher in <40% PR (33.3%) than in >40% PR group (8.1%, p < .05). In conclusion, the PR examination in semen can contribute to evaluate the reproductive performance of ram that will provide valuable insights into the semen evaluation.

Inverse relationship between autophagy and CTSK is related to bovine embryo quality.

Improving the quality and the developmental competence of in vitro produced (IVP) embryos is an indispensable goal for assisted reproductive technology. Autophagy is a major protective mechanism for intracellular degradation of unnecessary cytoplasmic components. Autophagy ends by the fusion between autophagic vacuoles and lysosomes, allowing the degradation of the cargo by lysosomal enzymes, especially the cathepsins (CTSs). However, it is still unclear how autophagy and cathepsin K (CTSK) relate to embryo development. This study evaluated (1.) the activities of autophagy and CTSK in relation to bovine embryo quality and (2.) the effect of autophagy induction and/or CTSK inhibition on preimplantation embryo development and quality. We show here that good-quality embryos exhibited a greater autophagic activity and less CTSK activity compared to poor-quality embryos. Blastomeres of an individual embryo may vary in their quality. Good quality blastomeres showed an increased autophagic activity and decreased CTSK activity compared to poor-quality blastomeres within the same embryo at different developmental stages. Importantly, induction of autophagy and/or inhibition of CTSK improved the developmental rate (increased blastocyst and hatching rates) and the quality (increased total cell number and decreased the percentage of apoptotic cells) of IVP bovine embryos. These results demonstrate a promising approach to selectively isolate good-quality embryos and improve the efficiency of IVEP of cattle embryos.

Kisspeptin injection improved the semen characteristics and sperm rheotaxis in Ossimi ram.

The aim of the present study was to investigate the effect of kisspeptin-10 (Kp10) injection on semen characteristics, testosterone (T) production and sperm rheotaxis using microfluidic devices in immature ram. Computer-assisted sperm analysis (CASA) with controlled flow velocity was used to explore the kinetic parameters of sperm and positive rheotaxis (PR %). PR % was defined as the number of PR sperms over the number of motile sperms. Healthy Ossimi rams were randomly divided into two groups; a saline-treated control group and Kp10-treated one (5 µg/kg body weight). Treatments were given by intramuscular injection once a week for 1 month. After 1 month, the semen was collected and evaluated weekly for 6 weeks, while the blood samples were collected every 2 weeks for the next 8 weeks. Semen properties were significantly affected by Kp10 injection (p < .01). The Kp10 increased the volume, sperm concentration and percentages of live sperm compared with those of control. Additionally, sperm trajectories and rheotaxis get improved by the injection of Kp10 with time. Furthermore, kisspeptin improved the secretion of testosterone levels throughout the period of study. In conclusion, injections of the Kp10 had a positive impact on semen characteristics as well as improved sperm rheotaxis of Ossimi rams in subtropics.

Experimental obesity and diabetes reduce male fertility: Potential involvement of hypothalamic Kiss-1, pituitary nitric oxide, serum vaspin and visfatin.

Reproductive dysfunction is a common consequence of both obesity and diabetes. This study investigated the impact of obesity and diabetes, alone or combined, on physiological reproductive parameters in male rats. Twenty-four male Wistar Albino rats were divided into four groups: Control; obese non-diabetic; diabetic; and obese diabetic. Obesity was provoked by consumption of a high-fat diet (HFD) consisting of 40% energy from fat for 90 days. Diabetes was induced by an intraperitoneal injection of streptozotocin at a dose of 40 mg/kg/day for three consecutive days. Semen, histopathological, and morphometric analyses were carried out. Serum testosterone, luteinizing hormone (LH), and vaspin and visfatin were measured using ELISA kits. Hypothalamic Kiss-1 mRNA was detected using qPCR and pituitary nitric oxide (NO) was determined using Griess reagent. Our results showed a decrease in semen quality parameters, testosterone, and LH levels with degenerative changes in the testes in experimental groups when compared to control group. This had a positive correlation with hypothalamic Kiss-1 and a negative correlation with pituitary NO and serum vaspin and visfatin. In addition, adverse effects were more pronounced in animals with obesity and diabetes combined compared to rats who were either diabetic or obese. In conclusion, obesity and diabetes, alone or combined, had a negative impact on male rat fertility. Moreover, obesity and diabetes combined had more harmful effects on male fertility when compared with obesity alone. Hypothalamic Kiss-1, pituitary NO, and serum vaspin and visfatin may play a role in the pathophysiology of male infertility-associated with obesity and diabetes.

Relationship between the antral follicle count in bovine ovaries from a local abattoir and steroidogenesis of granulosa cells cultured as oocyte-cumulus-granulosa complexes.

The antral follicle count (AFC) is used as an indicator of cow fertility. We herein investigated the relationship between AFC and the steroidogenesis of granulosa cells and confirmed the developmental competence of oocytes derived from early antral follicles (0.5-1.0 mm) using in vitro growth culture. Slaughterhouse-derived ovaries were divided into high (≥ 25) and low (< 25) AFC groups based on AFC (≥ 2.0 mm). Oocyte-cumulus-granulosa complexes (OCGCs) collected from early antral follicles were cultured for 12 days. The total number, viability, and diameter of granulosa cells and estradiol-17ß and progesterone production during the culture were evaluated. Surviving oocytes on day 12 were subjected to in vitro maturation, and their volume and nuclear status were evaluated. Some oocytes were subjected to the evaluation of developmental competence to blastocysts. Although the total number and viability of granulosa cells did not differ between the groups, granulosa cell diameters were smaller in the high AFC group than in the low AFC group. The estradiol-17ß and progesterone ratio on day 8 was higher in the high AFC group than in the low AFC group. Oocyte volumes and nuclear maturation rates were greater in the high AFC group than in the low AFC group. The development rate to blastocysts was 9.1% in the high AFC group, while no oocytes developed to blastocysts in the low AFC group. Therefore, estradiol-17ß production by granulosa cells appears to be greater in high AFC cattle than in low AFC cattle, thereby promoting the acquisition of oocyte competence.

Effects of pre-maturational culture duration on developmental competence of bovine small-sized oocytes.

We investigated the effects of pre-maturational (pre-IVM) culture on the developmental competence of small-sized bovine oocytes (110 and < 115 µm). Oocytes were cultured with 3-isobutyl-1-methylxanthine (IBMX) for 0, 5, or 10 h and subjected to in vitro maturation, fertilization, and culture. The cleavage rate (73%) of small-sized oocytes with 5 h pre-IVM was higher than those with 0 and 10 h pre-IVM (61 and 62%, respectively). The blastocyst rate (16%) of embryos derived from small-sized oocytes with 5 h pre-IVM was higher than those with 0 and 10 h pre-IVM (9 and 8%, respectively). In addition, small-sized oocytes with 5 h pre-IVM had a higher mean cell number in blastocysts (134.1 ± 34.8) than those with 0 and 10 h pre-IVM (100.2 ± 17.2 and 107.8 ± 23.7, respectively). In conclusion, the pre-IVM of small-sized oocytes with IBMX for 5 h improved the developmental competence of bovine oocytes, as well as the quality of blastocysts.

The protective role of folic acid against testicular dysfunction in lead-intoxicated rat model.

There is an increasing concern over male reproductive toxicity caused by lead exposure. Folic acid (FA) is supposed to be a promising therapeutic strategy against lead toxicity. Therefore, the aim of this experimental study was to shed light on the potential protective role of FA on lead-induced testicular dysfunction in rats and its possible underlying mechanistic pathways. Rats (n = 24) were divided into four groups: Control, FA, Lead, and FA+Lead group. After 4 weeks, lead intoxication resulted in a marked reduction in the relative testicular weight and the serum level of testosterone, an impairment in the characters of semen analysis, and an increased content of lead, malondialdehyde and both interleukin-6 and -10 and a decreased antioxidant enzyme levels in the testicular tissue homogenate. Furthermore, marked degenerative histological changes and an increased expression of NF-κB were also noticed in the testicular tissue of Lead group. Supplementation of FA in association with lead considerably alleviated these adverse outcome responses most probably owing to its cytoprotective ability as emerged from combating the oxidative stress and inflammatory reactions. We concluded that FA could act as a highly effective fighting approach against lead-associated testicular toxicity.

Effect of bovine cumulus-oocyte complexes-conditioned medium on in-vitro maturation of canine oocytes.

PURPOSE: To investigate the ability of medium conditioned with bovine cumulus-oocyte complexes (COCs) to support nuclear maturation of canine oocytes recovered from domestic dog ovaries. METHODS: Cumulus-oocyte complexes were obtained from ovaries of domestic bitches (8 months old to 7 years old), and in-vitro maturation was evaluated in TCM-199 supplemented with different concentrations (0, 20, 30 or 50%) of bovine COCs-conditioned medium (BCM). The canine COCs were cultured for 72 or 96 h at 38.5°C in 5% CO2, 5% O2 and 90% N2. The bovine COCs-conditioned medium was obtained from culture of bovine COCs with TCM-199 supplemented with 5% FCS for 22 h at 38.5°C in 2% CO2, 98% air. RESULTS: The proportion of germinal vesicle breakdown (GVBD) after 72 h was significantly higher (P < 0.05) in medium supplemented with 30% BCM (20.7%) compared with the control group (13.4%). The rates of GVBD-MII stage were significantly higher (P < 0.05) when oocytes were matured with BCM at concentration of 30% (41.5%) compared with control (26.6%) after 72 h in-vitro culture. After 96 h in-vitro culture, the oocytes matured in medium supplemented with 30% BCM (5.5%) showed a significant increase (P < 0.05) in the proportion of MII compared with control (0.7%). However, increasing the cultivation time from 72 to 96 h resulted in an increase in oocyte degeneration rate. CONCLUSIONS: The results suggested that bovine COCs-conditioned medium supplementation significantly increased nuclear maturation of canine oocytes.