Plasma Testosterone Concentrations Research Articles

Plasma steroids and immune measures vary with restraint duration in a toad (Rhinella icterica)

Immunoenhancing effects have been widely described following acute stressors in several vertebrates, and valuable contributions have been made from studies on acute stress to understand hormonal-immune interactions. However, most studies focus on hormonal and immune responses after standardized time lapses, neglecting potential influence of duration of exposition to stressor. Herein, we investigate fluctuations of plasma hormone concentrations (corticosterone and testosterone) and immunity (neutrophil to lymphocyte ratio, phagocytosis of blood cells, and plasma bacterial killing ability) in a toad species (Rhinella icterica) in response to six different periods of exposure to restraint stress. We observed increased plasma corticosterone concentrations following restraint in all sampled times (0.5 to 48 h), with the highest values being observed during the first hour (0.5 to 1 h). Restraint-induced increases in the neutrophil to lymphocyte ratio and phagocytosis percentage were observed from the first 0.5 h, gradually increasing after that with the time of restraint. We also observed decreased testosterone plasma concentrations in response to a more prolonged restraint (24 and 48 h). No changes were observed in plasma bacterial killing ability following restraint. Together, our results demonstrate dynamic time-related hormonal and immune changes. These results point to the fact that for some species measuring hormonal and immune variables at single time points following a stressor might work better when preceded by a study of the temporal changes of the response variables to the stimuli applied. Also, time of response needs to be considered when different variables are used as proxies of stress.

Chronic exposure to sublethal concentrations of saxitoxin reduces antioxidant activity and immunity in zebrafish but does not affect reproductive parameters

Saxitoxin produced by dinoflagellates and cyanobacteria can be transferred to humans through intoxicated organisms such as fish, but limited research has addressed the adverse effects of this toxin on aquatic organisms. In this study, we measured the potential effects of a 90-day exposure to saxitoxin (0.1 or 1 µg·L - 1) on body weight and length, antioxidant defense system, immunity, sex hormones, and genes involved in associated key metabolic pathways in zebrafish (Danio rerio). Significant impairments in body weight and length were observed in response to 1 µg·L - 1 saxitoxin in both male and female zebrafish. A significant increase in the levels of malondialdehyde, together with decreased enzymatic activities of catalase and superoxide dismutase, was observed in fish of both sexes exposed to 1 µg·L - 1 saxitoxin, indicating the occurrence of lipid peroxidation and oxidative stress. Immune parameters such as alternative complement activity, lysozyme activity, and immunoglobulin content were also significantly reduced. However, exposure of male and female zebrafish to saxitoxin for 90 days did not significantly affect reproductive parameters such as the gonadosomatic index and plasma concentrations of vitellogenin, estradiol, and 11-keto testosterone. Transcriptional responses showed similar trends to those of the biochemical parameters, as genes involved in the antioxidant defense system and immunity were downregulated, whereas the transcription of genes related to reproductive metabolism showed no significant change upon treatment with 1 µg·L - 1 saxitoxin. Our findings indicate that long-term exposure to a sublethal concentration of saxitoxin can inhibit growth through induction of oxidative stress and immunosuppression, while the reproductive parameters of zebrafish are not a main target of this toxin at sublethal concentrations.

Effects of gonadectomy on clinical-hematological, metabolic and hormone conditions of cockerels

Gonadectomy (caponization) of cockerels is an important way of increasing their productivity, improving the quality and taste properties of the meat, and obtaining dietary products. The objective of the study was the influence of gonadectomy of cockerels on their clinical condition, morphological composition of blood, hemoglobin content and anabolism in the tissues, their productivity and chemical content of the muscles. In the experiment, we used 30 Adler silver cockerels aged 4 weeks, which were divided into two groups: control (intact) and experimental (gonadectomized) according to the analogue principle, with 15 individuals in each. The experiment lasted for 185 days, including the main period of 175 days. We determined that the body temperature of cockerels after gonadectomy increased as early as days one and three. In the blood of gonadectomized cockerels, the number of leukocytes decreased by 36.3%, thrombocytes – by 24.7%, while hemoglobin concentration, numbers of heterophils, eosinophils, basophils, monocytes and lymphocytes did not change compared with the intact individuals. On days 20 and 185 of the experiment, the concentration of testosterone in the blood plasma of gonadectomized cockerels was lower respectively by 57.1% and 53.1%, whereas no differences were found prior to gonadectomy and on the third day of the experiment. We determined increase in uric acid concentration and decrease in the level of ionizing calcium in blood plasma of gonadectomized cockerels compared with intact individuals, while the content of cortisol, glucose, total protein, triglycerides, cholesterol, total calcium and inorganic phosphorus, and also activities of alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase did not change. Live weight of gonadectomized cockerels in the period from 45 to 105 days of the raising did not change, and was higher by 10.4–17.0% from day 115 to 145, and further remained not different from the control. Thoracic muscles of gonadectomized cockerels contained 1.8 times more fat, while the levels of moisture, dry matter, protein and ash in thoracic and thigh muscles did not change. The conducted studies have revealed the effect of gonadectomy of roosters on clinical-hematological parameters, metabolic processes, productivity of birds and chemical composition of muscles and may be used to improve the production of chicken meat with high dietary and taste properties.

A semi-extensive management system reduces plasma testosterone concentrations, sexual behaviour and sperm production in male goats from subtropical latitudes

Context In subtropical latitudes, most goats are managed in semi-extensive management systems, eating only available vegetation. Underfeeding is common during the dry season and may reduce the endocrine and sexual activities of male goats. Aims The aim of the study was to determine plasma testosterone concentrations, sexual behaviour and sperm production of male goats kept in a semi-extensive management system for 12 months, from March to February. Methods Control males were placed indoors and were well nourished (n = 5). Experimental males were placed in a flock and grazed daily with females for 7 h, eating only natural vegetation (n = 5). Experimental males were fitted with an apron to prevent mating during the study. Plasma testosterone concentrations were determined every 2 weeks. Sexual behaviour and sperm production were determined in May and June (beginning of breeding season), August and October (middle of season), and November and December (end of season). Key results Plasma testosterone concentrations were higher (P < 0.05) in control than experimental males in June and from August to December. The percentage of males refusing to ejaculate was lower (P < 0.05) in control than experimental males throughout the study. Total number of spermatozoa per ejaculate was higher (P < 0.05) in control than experimental males in May, June, November and December. Sperm progressive motility was higher (P < 0.05) in control than experimental males in May and June and from October to December. Conclusions The results indicate that male goats in a semi-extensive management system have lower plasma testosterone concentrations, sexual behaviour, and sperm quantitative and qualitative production than those in a confined management system. Implications Nutritional supplementation may improve the endocrine and sexual activity of male goats in semi-extensive management systems.

Polymorphisms of the melatonin receptor 1A (MTNR1A) gene do not affect sexual activity, plasma testosterone concentrations or testicular characteristics of 8-month-old ram-lambs born in autumn

Thirty-nine autumn-born ram-lambs were used to study whether polymorphisms of the melatonin receptor 1A (MTNR1A) gene influence some reproductive parameters of Oct-born ram-lambs at 8 months of age. Animals were genotyped for their RsaI (CC, n=24; CT, n=6; TT, n=9) and MnlI (GG, n=27; GA, n=6; AA, n=6) allelic variants of the MTNR1A gene. Liveweight (LW) was recorded bi-weekly until Jun, and scrotal perimeter (SP) was recorded once per month. From mid-Jan, blood samples were collected weekly to measure plasma testosterone concentrations. In mid-Jun, individual serving-capacity tests were performed. Testicular ultrasonography was performed at the end of the experiment. The effects of the week and gene polymorphism on LW, scrotal perimeter, and plasma testosterone concentrations were evaluated statistically by the GLM for repeated measures procedure. LW, SP, and plasma testosterone concentrations were significantly (P<0.001) affected by week, but neither of the polymorphisms of the melatonin receptor 1A (MTNR1A) gene affected these variables. In the serving tests, genotypes did not differ significantly in individual behaviors or the number of events (CC: 24.1±3.2, CT: 26.3±13.0, TT: 16.8±4.8, GG: 22.0±3.0, GA: 21.0±6.7, AA: 27.8±12.7). Testicular features assessed by ultrasonography did not differ significantly among genotypes. In conclusion, the polymorphisms of the melatonin receptor 1A (MTNR1A) gene did not have an effect on the reproductive characteristics of autumn-born ram lambs, based on serving capacity tests, testosterone secretion, and testicular measurements at 8 months of age.

Dose-Dependent Degeneration of Leydig Cells Following Kisspeptin-10 Administration: An Ultrastructural Study.

The discovery of kisspeptin signaling as a key regulator of gonadotropin- releasing hormone (GnRH) secretion from the hypothalamus enhanced our understanding of the neuroendocrine regulation of mammalian reproduction. Effects of central and peripheral administration of kisspeptin on plasma gonadotropins, testosterone, and spermatogenesis are studied in detail. The present study was conducted to check the ultrastructure of Leydig cells in prepubertal male rats in response to the administration of a range of kisspeptin doses. We administered a range of kisspeptin-10 doses (1 μg, 1 ηg, and 10 ρg) intraperitoneally to prepubertal male Sprague-Dawley rats (PND 35) twice daily after every 12 hours. Control rats were injected with physiological saline in parallel. At the end of the treatment, plasma concentrations of testosterone were measured by competitive binding radioimmunoassay, and small pieces of rat testicular tissue were processed for electron microscopy to examine the ultrastructure of Leydig cells. Plasma testosterone concentration was reduced significantly at 1ηg (P<0.05) and 1μg (P<0.01) doses as compared to control. Distinct ultrastructural changes categorized as dilatation of cytoplasmic organelles, irregularly shaped nuclei with nuclear membrane invaginations, reduced nuclear sizes, degeneration, and vacuolation were observed in the kisspeptin-10 treated Leydig cells as compared to control. Quantification of the data showed reduced Leydig cell indices and hyperplasia of the interstitial cells. It is concluded that chronic intermittent administration of kisspeptin-10 has a dose-dependent degenerative effect on the plasma testosterone levels and Leydig cells ultrastructure in prepubertal male rats.

Plasma profile of follicle-stimulating hormone and sex steroid hormones after a single epidural administration of follicle-stimulating hormone via caudal vertebrae in Holstein dry cows.

The conventional follicle-stimulating hormone (FSH) treatment for bovine superstimulation involves multiple intramuscular injections, which is stressful for animals and onerous. We herein investigated whether a single epidural injection of porcine FSH (pFSH) can induce superovulation and peripheral concentrations of pFSH and steroid hormones after the treatment in Holstein dry cows. We intramuscularly administered pFSH twice daily to three cows for 3 days (control) or a single epidural pFSH administration (epidural). Numbers of follicles (≥10mm in diameter) at estrus and corpora lutea at luteal phase were counted by ultrasonography. Blood was sampled from 0 to 104 h after the first pFSH administration and plasma pFSH, progesterone, androstenedione, testosterone, and estradiol-17β concentrations were measured. Numbers of follicles (control: 18.3 ± 7.5, epidural: 15.7 ± 4.0; mean ± SD) and corpora lutea (control: 7.3 ± 4.2, epidural: 8.0 ± 2.6) were similar between both treatments. Plasma pFSH concentrations were higher in epidural than in control (p < 0.01). Although no significant differences were observed in progesterone, androstenedione, or estradiol-17β concentrations between the groups, testosterone concentrations were slightly lower with the epidural treatment than with the control treatment (p = 0.08). In conclusion, superovulation was induced by a single epidural injection of pFSH, which achieved higher pFSH level than the multiple injections in Holstein dry cows.

Investigation of pathology associated with Chlamydia pecorum infection in the male reproductive tract, and the effect on spermatogenesis and semen quality in the koala (Phascolarctos cinereus)

There is growing evidence that Chlamydia pecorum infection of the male koala reproductive tract causes inflammation and pathology of the urogenital tract. Previous studies have revealed that male koalas exhibiting severe clinical signs of urogenital chlamydial disease had an increased incidence of sperm DNA fragmentation and abnormal sperm morphology, suggestive of chronic exposure to C. pecorum infection and/or inflammation in the testis and epididymis, with residual pathology and lesions disrupting spermatogenesis and maturation of spermatozoa. This study specifically aimed to determine whether pathology associated with chlamydial infection in different regions of the male koala reproductive tract had an adverse effect on classical seminal parameters, sperm DNA quality and endocrine function (testosterone secretion) of naturally infected males. Semen from 58 sexually mature male koalas deemed not suitable for rehabilitation or treatment was assessed, in addition to undertaking a GnRH challenge to determine the androgenic capacity of the testis. Following euthanasia, tissue samples from testes, epididymis and prostate were evaluated for histopathology and real time polymerase chain reaction (qPCR). A significant difference in sperm concentration was observed between males with unilateral and bilateral testicular atrophy and C. pecorum infection (P = 0.011); and between males with unilateral atrophy and C. pecorum infection in one testis and bilateral normal testes with no C. pecorum infection (P = 0.008). No significant association was found for any other semen parameters when categorised by histopathology and C. pecorum tissue presence within the testes, epididymis and prostate. Plasma testosterone concentrations did not significantly differ between testicular histopathology diagnosis and/or C. pecorum infection status. This study suggests Chlamydia infection and inflammation may not be the predominant reason of disruption to spermatogenesis in the wild koala but rather testicular degeneration and atrophy, irrespective of Chlamydia infection, appears to be the primary reason of decreased sperm concentration.

Chronic Dietary Exposure of Roosters to a Glyphosate-Based Herbicide Increases Seminal Plasma Glyphosate and AMPA Concentrations, Alters Sperm Parameters, and Induces Metabolic Disorders in the Progeny.

The effects of chronic dietary Roundup (RU) exposure on rooster sperm parameters, fertility, and offspring are unknown. We investigated the effects of chronic RU dietary exposure (46.8 mg kg−1 day−1 glyphosate) for 5 weeks in 32-week-old roosters (n = 5 RU-exposed and n = 5 control (CT)). Although the concentrations of glyphosate and its main metabolite AMPA (aminomethylphosphonic acid) increased in blood plasma and seminal fluid during exposure, no significant differences in testis weight and sperm concentrations were observed between RU and CT roosters. However, sperm motility was significantly reduced, associated with decreased calcium and ATP concentrations in RU spermatozoa. Plasma testosterone and oestradiol concentrations increased in RU roosters. These negative effects ceased 14 days after RU removal from the diet. Epigenetic analysis showed a global DNA hypomethylation in RU roosters. After artificial insemination of hens (n = 40) with sperm from CT or RU roosters, eggs were collected and artificially incubated. Embryo viability did not differ, but chicks from RU roosters (n = 118) had a higher food consumption, body weight and subcutaneous adipose tissue content. Chronic dietary RU exposure in roosters reduces sperm motility and increases plasma testosterone levels, growth performance, and fattening in offspring.

Inter-annual repeatability and age-dependent changes in plasma testosterone levels in a longitudinally monitored free-living passerine bird.

While seasonal trends in testosterone levels are known from cross-cohort studies, data on testosterone inter-annual individual repeatability in wild birds are rare. Also, our understanding of hormonal age-dependent changes in testosterone levels is limited. We assessed plasma testosterone levels in 105 samples originating from 49 repeatedly captured free-living great tits (Parus major) sampled during the nesting to investigate their relative long-term repeatability and within-individual changes. Furthermore, we examined the inter-annual repeatability of condition-related traits (carotenoid- and melanin-based plumage ornamentation, ptilochronological feather growth rate, body mass, and haematological heterophil/lymphocyte ratio) and their relationships to testosterone levels. We show that testosterone levels are inter-annually repeatable in females, with a non-significant pattern in males, both in absolute values and individual ranks (indicating the maintenance of relative status in a population). In males, we found a quadratic dependence of testosterone levels on age, with a peak in midlife. In contrast, female testosterone levels showed no age-dependent trends. The inter-annual repeatability of condition-related traits ranged from zero to moderate and was mostly unrelated to plasma testosterone concentrations. However, males with elevated testosterone had significantly higher carotenoid-pigmented yellow plumage brightness, a trait presumably involved in mating. Showing inter-annual repeatability in testosterone levels, this research opens the way to further understanding the causes of variation in condition-related traits. Based on a longitudinal dataset, this study demonstrates that male plasma testosterone undergoes age-related changes that may regulate resource allocation. Our results thus suggest that, unlike females, male birds undergo hormonal senescence similar to mammals.

Vitrification of Iberian wolf (Canis lupus signatus) sperm: A possible alternative to conventional cryopreservation

Sperm vitrification is a simple, inexpensive method that allows the cryopreservation of sperm in the field and for endangered species is a useful alternative to conventional freezing. The study, therefore, is focused on the suitability of vitrification for cryopreserving Iberian wolf sperm and utilizing plasma testosterone concentration as a marker for procedure efficacy. Sperm and blood samples were collected from 17 wolves. There were 14 samples suitable for cryopreservation (12 ejaculated and two epididymal). Immediately after collection, these samples were proportioned into two aliquots for conventional freezing using a Tris-citric acid-glucose based extender (TCG) or vitrification utilizing an animal protein free extender (HTF®). Vitrification occurred by directly plunging a sperm suspension into liquid nitrogen. Sperm were assessed for motility, membrane integrity, acrosomal status and DNA integrity before and after cryopreservation. With both techniques, there were similar post-thaw/warming results (P > 0.05) with respect to progressive motility, kinetic variables VCL, VSL, VAP and BCF, DNA fragmentation, sperm membrane functionality and morphological abnormalities. Total motile sperm, progression ratios LIN, STR, and WOB, the ALH, sperm viability and sperm with intact membrane and acrosome were greater (P < 0.05) in the conventional frozen-thawed sperm than vitrified-warmed sperm. Plasma testosterone concentrations varied from 0.0 ng/mL to 7.7 ng/mL. For epididymal sperm, sperm motility and viability following thawing were greater in vitrified-warmed samples than conventionally-frozen samples; however, small sample numbers precluded statistical analysis. When considered together, these results indicate vitrification may be a possible alternative for wolf sperm cryopreservation.

Ageing affects plasma steroid concentrations and testicular volume, echotexture and haemodynamics in rams.

The objective was to determine the effect of ageing on plasma steroid concentrations and testicular volume, echotexture and haemodynamics in Ossimi rams. Twenty-four rams were allocated, by age, into three groups: young (1year; 32.00±0.78kg, n=6); mature (2.5years; 48.00±1.02kg, n=9) and aged (7.5years; 65.00±1.10kg, n=9). Aged rams had the highest (p˂0.05) plasma testosterone concentrations 3.90±0.75ng/ml (three-fold the other groups), whereas in young rams, plasma estradiol-17β concentrations (5.85±0.34pg/ml) were approximately one-quarter (p˂0.05) of other groups. Regarding pulsed-wave Doppler indices values of the (resistive index RI; and pulsatility index PI) were lower (p˂0.05) in the mature (0.32±0.04 and 0.36±0.03, respectively) and aged (0.51±0.03 and 0.77±0.06) rams compared to young rams (0.62±0.03 and 0.98±0.07). In the young group, testicular pixel intensity was highest (p˂0.05) but values of coloured area/pixels were lowest (p˂0.05) compared to the other groups. In conclusion, these parameters indicate that aged rams (7.5years) are likely to be suitable for breeding ahead of the younger aged groups (1 and 2.5years) examined, but a final decision would need to be tempered by consideration of other parameters in the breeding soundness examination (BSE), such as physical soundness and semen characteristics. Of the parameters studied, testicular pixel intensity could be most readily incorporated in a BSE for deciding a ram's breeding efficiency.

Systematic review and meta-analyses on associations of endogenous testosterone concentration with health outcomes in community-dwelling men

ObjectivesThe overall study aim is to clarify the relation of endogenous sex hormones with major health outcomes in men. This paper reports a systematic review focusing on published estimates for...

Sexually inexperienced, photo-stimulated, 27-month-old male goats showed undiminished sexual behavior and ability to induce estrus and ovulation in anestrous females

Sexually inexperienced male goats aged 8–21 months displayed relatively low sexual behavior when exposed to females for the first time. We aimed to determine whether i) photo-stimulation can overcome sexual inexperience in 27-month-old male goats, ii) sexually inexperienced, photo-stimulated male goats aged 27 months display the same sexual behavior as experienced goats, and iii) sexually inexperienced and experienced photo-stimulated male goats induce estrous behavior and ovulation in anestrous female goats. Five sexually inexperienced and five sexually experienced males were subjected to artificially long days (16 h of light and 8 h of darkness per day) for 2.5 months and then were exposed to females at 27 months of age (n = 50/group) for 15 days. Plasma testosterone concentrations did not differ between the two groups (P > 0.05). Sexual behavior was recorded on days 0, 1, 2, and 3 after introduction to the female groups. The inexperienced group displayed more nudging on day 2 than on days 0, 1, and 3 (P < 0.05), and both experienced and inexperienced males recorded more anogenital sniffing on days 0 and 3 than on days 1 and 2 (P < 0.05). Both experienced and inexperienced males made more mounting attempts on days 0 and 2 (P < 0.05), and experienced males made more mounts with intromission on day 3 (P < 0.05). The interval between the introduction of males to females and first estrus, estrous behavior, ovulation, and pregnancy rate were similar in both inexperienced and experienced males (P > 0.05). In conclusion, sexually inexperienced or sexually experienced photo-stimulated males aged 27 months displayed high sexual behavior and stimulated estrous behavior and ovulation in anestrous female goats.

Melatonin along with eCG improves fresh semen quality and plasma concentrations of melatonin and testosterone during non-breeding season in Beetal bucks

Under subtropical regions, the breeding activities of goats are restricted to a specific season. Several hormonal regimens have been used to improve reproductive performance. However, different hormonal protocols have certain limitations. The effect of melatonin + eCG therapy on fresh semen, plasma testosterone, and melatonin profiles of bucks was evaluated during the non-breeding season. Nine bucks were assigned to three treatments (control; normal saline, melatonin alone, and melatonin + eCG; n = 3 in each group). Except for control, all bucks were implanted melatonin subcutaneously and eCG was administered every fourth day to melatonin + eCG treated bucks. Weekly two fresh ejaculates per buck for 10 weeks (n = 180) were evaluated for sperm concentration, motility, kinematics (VCL, VSL, VAP, and ALH), non-viability, morphological abnormality, DNA fragmentation/damage, acrosomal and mitochondrial integrity. Weekly plasma profiles of testosterone and melatonin were monitored. From the 4th week onward, non-viable and morphological abnormal sperms and decreased (P < 0.05) while sperm progressive motility and kinematics, acrosomal and DNA integrity improved (P < 0.05) in melatonin + eCG than melatonin alone and control treatments. The concentrations of testosterone (ng/mL) increased (P < 0.05) from the 3rd week onward in melatonin + eCG (4.2 ± 0.2) than melatonin alone (1.2 ± 0.1) and control treatments (0.28 ± 0.1). An increase (P < 0.05) in the concentration of melatonin (pg/mL) was observed at 5th week in melatonin + eCG (12.5 ± 0.2) than melatonin alone (10.2 ± 0.1) and control treatments (3.89 ± 0.2); however, by 9th-week melatonin alone and melatonin + eCG had similar melatonin profiles. Compared to other treatments, melatonin + eCG therapy quickly improves and sustains fresh semen quality and plasma testosterone profile of bucks during the non-breeding season.

Measuring the Effects of a Single Dose of Human Chorionic Gonadotropin (hCG) on Plasma Testosterone Concentrations in Leopard Geckos (Eublepharis macularius)

Current estimates suggest that one-fifth of all reptiles are currently threatened with extinction because of anthropogenic activity, and that if nothing is done to mitigate these impacts, massive extinctions will occur over the coming decades. Assisted reproductive technologies (ART) can be used to develop ex vivo conservation programs to ensure species survival until environmental deficiencies can be corrected. Geckos represent one of the most diverse groups of reptiles on earth and need protection; however, to date, no ART have been developed for these animals. Exogenous human chorionic gonadotropin (hCG) is routinely used to alter reproductive hormones of vertebrates and are integrated in ART. The purpose of this study was to determine if a single dose of hCG could increase plasma testosterone concentrations in the leopard gecko (Eublepharis macularius) within 24 h of administration. The study was performed in September, which is considered the nonbreeding season for this species in the northern hemisphere. Twelve captive-bred, adult, male leopard geckos were randomly divided into three treatment groups and administered an intramuscular injection of hCG (50 or 100 IU) or saline. Whole blood was collected prior to injection (baseline) and 12 and 24 h after injection. Plasma testosterone concentrations were measured using an enzyme immunoassay kit. There was no significant difference (P = 0.195) in testosterone concentrations between treatment groups. Because there was no difference, an observed testosterone range (61.0 ± 42.5, 5.5–149.0) for September was calculated. These results suggest that captive leopard geckos may be in a recrudescence phase in September in the northern hemisphere.

Initial Characterization of Male Southern Stingray (Hypanus americanus) Reproductive Parameters and Preliminary Investigation of Sperm Cryopreservation.

Simple SummaryUnderstanding the reproductive biology of a species is critical for the development of biobanks and assisted reproductive techniques to aid in the genetic management of isolated populations. Male southern stingray (Hypanus americanus) reproductive examinations were opportunistically conducted in March and June. Semen and plasma were collected to characterize ejaculate parameters and to investigate the effect of plasma total testosterone on semen quality. Semen was used for preliminary sperm cryopreservation studies. Changes in semen quality were observed with changes in plasma testosterone concentrations and body conditions. Southern stingray spermatozoa were highly sensitive to cooling rates with slower rates, producing a higher post-thaw survival.This study investigated the reproductive biology and sperm cryopreservation of ex situ southern stingrays (Hypanus americanus) by semen collection and characterization and the development and validation of an enzyme-linked immunoassay for plasma total testosterone. Semen was collected in March and June using a manual massage technique, and the ejaculates were assessed for volume, pH, osmolarity, motility, status (0–5 scale: 0 = no forward progression, 5 = rapid linear progression) and total sperm count. Semen was extended in Hank’s elasmobranch ringer solution containing 10% DMSO, 10% glycerol or 5% glycerol with 5% N-methylformamide and cryopreserved using a conventional freezing method (~−50 °C/min) or a modified slow freezing method (~−3 °C/min). Body condition was scored from 1–5 and was noted to be low in March (1.93 ± 0.07) due to feeding practices and increased by June (2.93 ± 0.05) after dietary corrections were made. A concomitant increase (p < 0.05) in plasma total testosterone concentration and sperm motility was noted between March (8.0 ± 7.2 ng/mL, 5.71 ± 2.77%) and June (97.3 ± 11.3 ng/mL, 51.4 ± 14.3%). Samples cryopreserved using a modified slow freeze method (~−3 °C/min) had higher post-thaw motility and plasma membrane integrity than conventionally cryopreserved samples. Data indicate that southern stingray sperm morphometrics adheres to those of other elasmobranch species and that a slow cooling rate may be an avenue of research to improve southern stingray sperm survival during cryopreservation.

Toxic masculinity in red blood cell units? Testosterone therapy in blood donors revisited.

FDA guidelines limit the use of blood from donors taking testosterone replacement therapy (TRT) to red blood cell (RBC) concentrates, whereas plasma and platelets are discarded. The purpose of this study is to bring awareness to above-average free testosterone concentrations in RBC units from TRT donors. We quantified the concentrations of free (bioavailable; pg/ml) and total (protein bound and free; ng/dl) testosterone in plasma (frozen within 24 h) and supernatants from 42-day stored leukocyte-reduced RBC units from 17 TRT male donors and 17 matched controls (no TRT). Total testosterone concentrations were determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Free testosterone concentrations were quantified in the same samples using equilibrium dialysis/LC-MS/MS. Plasma free and total testosterone concentrations in TRT donors were 2.9 and 1.8 times higher than that of controls. Total testosterone concentrations in RBC supernatants were about 30% of that of plasma. In contrast, free testosterone concentrations in RBC supernatants were 80%-100% of that of plasma and were significantly (p=.005) higher in TRT compared with controls (252.3 ± 245.3 vs. 103.4 ± 88.2pg/ml). Supraphysiological free testosterone concentrations (>244 pg/ml) in RBC supernatants were observed in five TRT donors and two control donors. RBC units from TRT donors may contain supraphysiological concentrations of free testosterone. This may be resolved by avoiding blood collections soon after testosterone dosing and by enhanced screening of TRT donors. These data establish a rationale for new studies and reexamination of the current guidelines concerning the utilization of blood components from TRT donors.

Pre-analytical factors affect the accurate measurement of testosterone concentrations in plasma and serum of goats.

Several pre-analytical factors may influence the accurate measurements of testosterone (T) and therefore, these factors must be a significant concern. This study aimed to examine the effects of 1) time of sample collection, 2) delay to centrifugation, 3) sample matrix types, and 4) device and duration of sample storage on the T concentrations. Blood samples were collected from 34 bucks of Kacang goats. For testing the effect of collection time, 12 pairs of morning and afternoon samples were collected. For testing the effect of delayed centrifugation, 24 samples were subjected to treatments: (i) centrifuged ⟨1 hour after collection (control group), (ii) centrifuged 6, 12, and 24 hours after collection (test groups). For testing the different sample matrix types, 10 samples were processed as serum and plasma. For testing the effect of sample storage device and duration, 60 samples were subjected to treatments: i) frozen at -20OC (control group), ii) stored in a cooler box, a styrofoam box, and a thermos-flask for two, four, and six days (test groups). T concentrations were measured using a validated testosterone ELISA kit. Concentrations of plasma testosterone (pT) from morning samples were significantly higher compared to afternoon samples (p⟨0.05). Delayed centrifugation for up to 24 h decreased significantly on pT concentrations (p⟨0.05). The concentrations of T from serum and plasma did not differ and showed a strong correlation (r=0.981). Storage device and duration affected the T concentrations compared to frozen samples (p⟨0.05) which T concentrations were stable for up to 4 days in a styrofoam box and a thermos-flask and up to 6 days in a cooler box. In conclusion, the measurement accuracy and stability of T concentrations in goats are affected by collection time, delay to centrifugation, and device and duration of storage.

Voluntary Exercise Attenuates Hyperhomocysteinemia, But Does not Protect Against Hyperhomocysteinemia-Induced Testicular and Epididymal Disturbances.

The hyperhomocysteinemia (HHcy) is toxic to the cells and associated with several diseases. Clinical studies have shown changes in plasma concentrations of Hcy after physical exercise. This study aimed to assess the effect of HHcy on testis, epididymis and sperm quality and to investigate whether voluntary exercise training protects this system against damage caused by HHcy in Swiss mice. In this study, 48 mice were randomly distributed in the control, HHcy, physical exercise, and HHcy combined with physical exercise groups. HHcy was induced by daily administration of dl-homocysteine thiolactone via gavage throughout the experimental period. Physical exercise was performed through voluntary running on the exercise wheels. The plasma concentrations of homocysteine (Hcy) and testosterone were determined. The testes and epididymis were used to assess the sperm count, histopathology, lipoperoxidation, cytokine levels, testicular cholesterol, myeloperoxidase, and catalase activity. Spermatozoa were analyzed for morphology, acrosome integrity, mitochondrial activity, and motility. In the testes, HHcy increased the number of abnormal seminiferous tubules, reduced the tubular diameter and the height of the germinal epithelium. In the epididymis, there was tissue remodeling in the head region. Ultimately, voluntary physical exercise training reduced plasma Hcy concentration but did not attenuate HHcy-induced testicular and epididymal disturbances.