Induction Of Spermiation Research Articles

Sperm parameters following hormonal induction of spermiation in an endangered frog [the alpine tree frog] (Litoria verreauxii alpina).

With global amphibian biodiversity rapidly declining, improving reproductive technology outcomes has become essential. Captive breeding programs have struggled because amphibian breeding physiology often requires specific environmental cues that reproductive technologies can circumvent. This study tests the efficiency of hormonal induction by evaluating sperm quality in the endangered Litoria verreauxii alpina . We assessed the effects of exogenous hormones - gonadotrophin-releasing hormone agonist (GnRH-a, Lucrin), and human chorionic gonadotrophin (hCG, Chorulon) - on sperm quality. Hormone induction with hCG showed high efficacy while GnRH-a yielded a low response in producing sperm. Sperm quantity was affected by time post injection, with the greatest quantity at 1h post injection. Sperm quality was also affected by time, where the sperm head size decreased by 11% at 7h post injection. Based on the results from this study, we recommend that that sperm be collected soon after induction, and not more than 4h post induction in L. v. alpina . More work needs to be completed before recommending an optimal hormone induction method and dose, but 120IU of hCG per male was successful for inducing spermiation. This study represents a useful starting point for developing assisted reproductive techniques for non-model organisms.

Gonadotropin induction of spermiation in Senegalese sole: Effect of temperature and stripping time

Treatments with homologous recombinant follicle-stimulating and luteinizing hormones (rFsh and rLh, respectively) are known to enhance spermatogenesis and sperm production in sole, but the response can be highly variable depending on the dose, duration and time of the year of the rFsh treatment. To further investigate the physiological effects of rFsh and rLh on sperm production in sole, here we examined the pattern of spermiation of F1 males, of approximately 450 g, treated with rFsh and rLh under controlled temperature. In an initial trial at 12 °C, males were weekly injected intramuscularly with 18 μg kg−1 rFsh over five weeks and subsequently treated with a single injection of 18 μg kg−1 rLh. Histological analysis indicated that the rFsh+rLh treatment increased gonad weight and stimulated spermatogenesis, and also enlarged the size of the seminiferous and efferent duct (ED) tubules, resulting in a doubling of sperm production with respect to the controls. Sperm counts in the ED and sequential stripping of males at 24, 48 and 72 h post rLh injection further revealed that only one batch of spermatids is recruited into spermatozoa (Spz) differentiation after a single rLh induction. A peak of sperm accumulation in the ED occurs at 48 h, coinciding with the upregulation of genes potentially involved in Spz maturation. In a second experiment, we tested the effect of two rFsh doses (10 or 18 μg kg−1) over five weeks as previously, followed by one rLh injection at 12 °C or 17 °C. The results confirmed that spermiation was the highest 48 h after rLh treatment at 12 °C, which was increased in a dose-dependent manner with the dose of rFsh previously supplied (from 0.36 to 0.95 × 109 Spz kg−1). However, sperm production elicited with the low rFsh dose was potentiated by ~3-fold (from 0.36 to 1.06 × 109 Spz kg−1) when the rLh treatment was given at 17 °C. These data suggest that in Senegalese sole sperm collection should be carried out at 48 h after rLh treatment, and that a low dose of rFsh at 12 °C is highly efficient for stimulating sperm production when rLh is administered at a temperature close to that occurring during maximum natural spermiation.

Induction of Spermiation in Sterlet Acipenser ruthenus by PLGA Microparticle Delivery with Sustained Alarelin Release.

Simple SummaryCultured sterlet Acipenser ruthenus males do not usually undergo spontaneous spermiation, and if any sperm is obtained without hormone treatment, it is generally of diminished quality. We compared efficacy of stimulation of spermiation with carp pituitary extract to that of 35 µg kg−1 or 200 µg kg−1 body weight gonadotropin-releasing hormone analogue in a sustained release system. Hormone treatments caused a significant increase in testosterone and 11-ketotestosterone, as well as induced spermiation. The delivery system based on poly (lactic-co-glycolic acid) microparticles with slow release of Alarelin at 35 µg kg−1 BW effectively induced spermiation, and was associated with extended sperm collection compared to carp pituitary treatment. The sustained delivery system offers an excellent option for spermiation induction in cultured sterlet, and possibly other sturgeon.Carp pituitary treatment versus poly (lactiac-co-glycolic acid) microparticles with slow release of Alarelin at 35 µg kg−1 or 200 µg kg−1 body weight to induce spermiation was compared in sterlet Acipenser ruthenus. All hormone treatments initially increased testosterone and 11-ketotestosterone, with a subsequent decline in testosterone but consistent high levels of 11-ketotestosterone at 48 and 72 h post-treatment. Spermiation did not differ between hormone-treated groups, and was not detected in controls receiving saline solution. Administration of the carp pituitary led to maximum sperm production 24 h post-treatment, followed by a decrease at 48 h post-treatment, with no sperm obtained at 72 h. The effect of Alarelin at 35 µg kg−1 bw and carp pituitary did not differ at 24 and 48 h post-treatment, whereas 200 µg kg−1 bw Alarelin was associated with significantly lower spermatozoon concentration 24 h post-treatment compared to carp pituitary, with no difference in milt volume. Higher relative sperm production was observed 48 h after injection of Alarelin at 200 µg kg−1 bw compared to carp pituitary. Spermatozoon motility was significantly higher in fish receiving Alarelin at 35 µg kg−1 bw than 200 µg kg−1 bw. The treatment with optimal effect on inducing spermiation was poly (lactic-co-glycolic acid) microparticles with slow release of Alarelin at 35 µg kg−1 bw.

Influence of Environmental Temperature and Hormonal Stimulation on the In Vitro Sperm Maturation in Sterlet Acipenser ruthenus in Advance of the Spawning Season

Simple SummarySperm maturation (acquisition of the potential for motility and fertilization by morphologically developed spermatozoa) in sturgeons is atypical of ray-finned fishes: it occurs outside the testes during the transit of testicular spermatozoa through the kidneys into the Wolffian ducts. We recently developed a method in which testicular spermatozoa of sterlet Acipenser ruthenus are matured in vitro when incubated in seminal fluid derived from Wolffian duct sperm. In this study, we explored whether in vitro maturation of testicular spermatozoa depends on the environmental temperature and/or hormonal stimulation of spermiation. We studied spermatozoa motility parameters after in vitro maturation of testicular sperm, concentrations of sex steroid hormones and testis morphology in sterlet males at different stages of male preparation for spawning with and without hormonal induction of spermiation. The obtained data suggest that the ability of testicular spermatozoa to be matured was not related to the environmental temperature, while hormonal stimulation was an absolute requirement for optimal in vitro maturation. The use of in vitro matured testicular spermatozoa might have considerable potential in aquaculture or conservation programs, which can be realized in cases of accidental death of valuable broodstock or failure to obtain Wolffian duct sperm of high quality.Sturgeon sperm maturation occurs outside the testes during the transit of testicular spermatozoa (TS) through the kidneys and the Wolffian ducts. A method of in vitro TS maturation in sterlet Acipenser ruthenus was used to investigate the effects of temperature and hormonal stimulation of spermiation on the ability of TS to complete this process. Spermatozoa motility parameters after in vitro maturation of testicular sperm, concentrations of sex steroid hormones and testis morphology were studied in three groups of sterlet: (1) after overwintering in ponds (OW), (2) adapted to spawning temperature (ST), and (3) adapted to spawning temperature with hormonal induction of spermiation (ST-HI). Blood plasma concentrations of testosterone, 11-ketotestosterone and 17,20β-dihydroxy-pregnenolone increased significantly after hormonal induction of spermiation (group ST-HI). In all groups, TS were not motile. After in vitro sperm maturation, motility was up to 60% only in group ST-HI. The data suggest that the ability of TS to be matured in vitro was not related to the environmental temperature, while hormonal stimulation of spermiation during the spawning season was an absolute requirement for optimal in vitro maturation.

Efficacy of hormone stimulation on sperm production in an alpine amphibian (Anaxyrus boreas boreas) and the impact of short-term storage on sperm quality

The Southern Rocky Mountain boreal toad (Anaxyrus boreas boreas) has disappeared from much of its range in the alpine regions of Central and Western North America, and restoration efforts are compromised by limited knowledge of this species’ reproductive biology. This study aimed to establish whether assisted reproductive techniques could be used to improve breeding output in captive boreal toads by determining the most effective concentration of human chorionic gonadotropin (hCG) for induction of spermiation and viability of sperm during cold storage. Male toads (n = 21) were treated with a Low (3 IU g-1), Medium (10 IU g-1), or High (15 IU g-1) concentration of hCG and spermic urine samples were collected over 24 hrs. Treatment effectiveness was evaluated by measuring the response rate, Total Motility (TM), Forward Progressive Motility (FPM), Quality of FPM (QFPM), and concentration. For short-term cold storage, spermic urine samples (n = 13) were stored at 4 °C for 14 days and sperm TM and FPM monitored daily. All treatments induced spermiation; however, a greater number of toads produced sperm in the Medium and High treatments compared to the Low. Overall, TM, FPM, QFPM and sperm concentration were similar across all three treatments, but variation existed in the timing and duration of peak sperm production. Sperm motility was maintained for up to 14 days in cold storage, although the quality slowly decreased over time. An effective reproduction strategy for the boreal toad will provide a means to improve captive breeding efforts and increase our understanding of the reproductive physiology of alpine Bufonids.

Repeated hormonal induction of spermiation affects the stress but not the immune response in pikeperch (Sander lucioperca)

Hormonal induction of spermiation, previously reported to be immunogenic in fishes, is a common hatchery practice in pikeperch, Sander lucioperca. The aim of the present study was to investigate the effects of repeated induction of spermiation in pikeperch, following application of either human chorionic gonadotropin (hCG) or salmon gonadoliberine analogue (sGnRHa) on sperm quality indices as well as on immune and stress response. Mature males of pikeperch (n = 7 per group) were stimulated twice with five days between injections of either hCG (hCG; 500 IU kg−1), sGnRHa (sGnRHa; 50 μg kg−1) or NaCl (control group; 1 ml kg−1) to assess spermatozoa motility with a computer-assisted sperm analysis (CASA) system. During second sampling, blood plasma was sampled for humoral innate immune (peroxidase and lysozyme activities, ACH50), stress (cortisol, glucose) and endocrine (testosterone) markers. In addition, the head kidney was dissected to assay the expression of several immune genes (such as il1, c3, hamp, tnf-α and lys genes). The results indicate that hormonal treatment significantly increased sperm production. Sperm sampled after the hormonal treatment maintained its quality throughout the study, regardless of the sampling time. However, it appears that the application of hCG induced elevated cortisol and glucose plasma levels compared to the control group. Almost all immune markers, except the relative expression of hepcidin (hamp gene), were unaffected by the two hormones applied. The results showed that the induction treatment of spermiation processes in pikeperch resulted in an important physiological stress response for which the intensity varied according to the hormonal agent used. However, this stress response (more profound following application of hCG) was weakly associated with innate immune functions. On the other hand, a significant negative correlation between the expression of several important immune markers (peroxidase activity, relative expression of c3 and il1 genes) and sperm quality indices indicates significant involvement of immune status on sperm quality. The results obtained shed light on immune-system-induced modifications to sperm quality. The data presented here highlight the need for careful revision of broodstock management and selection practices where welfare status as well as individual predispositions of fish to cope with the stress should be taken under the consideration.

DNA methylation stability in fish spermatozoa upon external constraint: Impact of fish hormonal stimulation and sperm cryopreservation.

Highly differentiated mature spermatozoa carry not only genetic but also epigenetic information that is to be transmitted to the embryo. DNA methylation is one epigenetic actor associated with sperm nucleus compaction, gene silencing, and prepatterning of embryonic gene expression. Therefore, the stability of this mark toward reproductive biotechnologies is a major issue in animal production. The present work explored the impact of hormonal induction of spermiation and sperm cryopreservation in two cyprinids, the goldfish (Carassius auratus) and the zebrafish (Danio rerio), using LUminometric Methylation Assay (LUMA). We showed that while goldfish hormonal treatment did increase sperm production, it did not alter global DNA methylation of spermatozoa. Different sperm samples repeatedly collected from the same males for 2 months also showed the same global DNA methylation level. Similarly, global DNA methylation was not affected after cryopreservation of goldfish spermatozoa with methanol, whereas less efficient cryoprotectants (dimethylsulfoxide and 1,2-propanediol) decreased DNA methylation. In contrast, cryopreservation of zebrafish spermatozoa with methanol induced a slight, but significant, increase in global DNA methylation. In the less compact nuclei, that is, goldfish fin somatic cells, cryopreservation did not change global DNA methylation regardless of the choice of cryoprotectant. To conclude, global DNA methylation is a robust parameter with respect to biotechnologies such as hormonal induction of spermiation and sperm cryopreservation, but it can be altered when the best sperm manipulation conditions are not met.

Out-of-season artificial reproduction of common dace (Leuciscus leuciscus L.) under controlled conditions

This study focused on the artificial reproduction of common dace Leuciscus leuciscus (L.) outside the reproductive season. The results indicate there is the possibility of initiating the reproducion in individuals of this before the natural spawning season. There could be induction of spermiation by altering the environmental conditions. For females, hormonal injections were necessary for induction of final oocyte maturation and ovulation. Generally, there were high percentages of spermiation (100%) and ovulation (87%–100%) as well as low mortality (0%–11% and 7%–13% for males and females, respectively) among the induced spawners. The greatest embryo survival occurred when Ovaprim (84.4%) or hCG (89.6%) was administered, although the latency time using hCG was at least twice as long compared to when other spawning agents were used (84–92 hrs and 30–44 hrs). The results from present study could be applicable for optimization of breeding stock numbers in aquaculture enterprises and in providing insights for conservation of L. leuciscus endangered populations.

Induction of spermiation using Ovaprim™ with topical gill method in the silver rasbora (Rasbora argyrotaenia)

The main obstacles on silver rasbora (Rasbora argyrotaenia) culture are having the limited number of broodstock and spawning depending on the season. The purpose of this study was to determine the effect of different dosage of Ovaprim™ induction by topical gill method to silver rasbora spermiation in order to continue the production out of its reproduction season with an optimum dose. A total of 30 male fish with a weight of 7.78 ± 0.20 g and length 4.11 ± 0.31 cm was used in this research. Topical gill treatments of Ovaprim™ were administered with following doses; 0.15 μl/g, 0.25 μl/g, 0.35 μl/g, 0.45 μl/g and 0.55 μl/g body weight. Milt volume, sperm concentration, sperm motility, and sperm viability parameters were observed in this study to understand the optimum dose of Ovaprim™ for male silver rasbora breeders. Spermiation induction of silver rasbora using Ovaprim™ with topical gill method has been successfully carried out, indicating an increase (P < 0.05) in milt volume, sperm concentration, sperm motility, and sperm viability. According to results a dose of Ovaprim™ is recommended to be used the 0.25 μl/g body weight in the spermiation induction of silver rasbora.

Effects of hCG and salmon gonadoliberine analogue on spermiation in the Eurasian perch (Perca fluviatilis)

This study analysed (i) the effect of human chorionic gonadotropin (hCG) and salmon gonadoliberine analogue (sGnRHa) on the effectiveness of induction of spermiation and (ii) the effect of latency time following the application of those spawning agents on the quantity and quality of the sperm of Eurasian perch, Perca fluviatilis, obtained during out-of-season spawning. For this study, pond-reared fish were used which had been acclimated to the controlled conditions. Three groups were distinguished which were treated with either saline (0.9% NaCl; control group), hCG (500 IU kg−1) or sGnRHa (100 μg kg−1). The fish were kept in a recirculating system at 12 °C throughout the study, during which sperm was collected every two days between the 2nd and 10th day following hormonal treatment. During the study, quantitative (e.g. sperm volume, total sperm production) and qualitative (measured with a computer-assisted sperm analysis system – i.e. CASA) parameters were monitored. The results of the study indicate that the hormonal treatment had a highly beneficial effect on the spermiation rate (100% in experimental groups from day 6 following injection) as well as quantity, which increased 50% in experimental groups (over 2200 × 109 of spermatozoa per kg of body weight) by day 4 following injection. For the sperm quality, both spawning agents tested had a rather positive effect, although sperm motility rate (MOT) was seen to be significantly reduced on day 10 following the application of hCG (MOT = 72.8% ± 8.1), which was not observed after the application of sGnRHa (minimum mean MOT 81.7% ± 6.1). The results clearly indicate that hormonal treatment had a positive effect on spermiation in Eurasian perch, most apparent from day 6 following injection, regardless of the hormonal agent used. Though application of sGnRHa allowed a high volume of high quality sperm to be stripped for two days longer (up to day 10 post-injection) compared to the application of hCG.

Comparative effects of human chorionic gonadotropin (hCG) and Ovaprim™ (sGnRHa + domperidone) on the reproductive characteristics of wild-caught male Longspine scraper, Capoeta trutta (Heckel, 1843)

The effectiveness of two different hormonal treatments or a saline control on sperm characteristics, plasma sex steroids and testes histology of male Longspine scraper, Capoeta trutta (Heckel, 1843) collected from the wild and maintained in captivity were tested. Fish injected with Ovaprim™ had significantly increased gonadosomatic index (GSI) and sperm volume, but had decreased spermatozoa concentration and spermatocrit compared with control. Fish injected with hCG had significantly decreased motility duration of their sperm, as well as decreased spermatozoa concentration and spermatocrit. Both treatments increased seminal plasma pH compared with control, while decreases in osmolality of seminal plasma occurred only in Ovaprim™-treated fish.Hormonal treatments altered the seminal plasma sodium (Na+), calcium (Ca2+), magnesium (Mg2+), glucose, phosphorus, urea, creatinine, alanine aminotransferase (ALT), and alkaline phosphatase (ALP) concentrations. However, neither treatment affected the seminal plasma potassium (K+), chloride (Cl−), uric acid, total protein, cholesterol, triglycerides, and aspartate aminotransferase (AST) concentrations.The concentration of 17,20β-dihydroxy-4-pregnen-3-one (DHP) in the plasma was significantly enhanced by Ovaprim™ treatment, but not by hCG. On the other hand, plasma testosterone (T) was significantly elevated by both treatments.Histological examination of the testes revealed stimulation of testicular maturation following treatment with Ovaprim™, with lobules observed that were filled exclusively with a large number of spermatozoa (spz). In summary, hormonal treatments stimulated steroidogenesis and spermiation in male C. trutta, with highest efficiency of the Ovaprim™ treatment. Therefore, it can be concluded that like many other cyprinids, dopamine inhibitory action was operating in C. trutta, thus requiring anti-dopaminergic and hormonal treatments to overcome this in order to maximize steroidogenesis and induction of spermiation. Statement of relevanceThe research I present in this paper is novel, as:•The effectiveness of exogenous hormones on male C. trutta reproduction under controlled conditions;•The effect of hormonal treatment on a wide range of seminal plasma compositions in C. trutta;•Valuable information which will assist commercial facilities in organizing and, spermiation success in this new aquaculture species.

Comparison of methods to improve induction of spermiation in wild-caught carp (Cyprinus carpio carpio), a threatened species from the Caspian Sea basin

Wild carp (Cyprinus carpio carpio) forms the basis of an important fishery in the Southern Caspian Sea Basin which is increasingly underpinned by the release of cultured juveniles. A significant bottleneck to hatchery-rearing of juveniles is the spermiation of male broodstock. Therefore, four approaches to improving spermiation were investigated. The effectiveness of two delivery methods for the sustained release of salmon gonadotropin releasing hormone analogue (sGnRHa; i.e., via intramuscular cholesterol pellet vs emulsion injection) on the spermiation success and duration, sperm quality and quantity over 14days in wild-caught carp were compared to single injection of sGnRHa with Pimozide® (Linpe method) or carp pituitary extract (CPE). The consequence of the spermiation treatments on resulting embryonic quality was evaluated for subsequent fertilization and hatching success from wild male carp (mean weight±S.D. 1021±112g). All hormonal treatments, except for Linpe method, led to 100% spermiation of treated fish compared to only 25% in the control with no hormone intervention. The duration of spermiation, as well as the various quantitative variables of the sperm and the mean total sperm production were all generally improved with the sustained hormone delivery compared with the acute treatments. The GnRHa-FIA was the most effective method for improving spermiation.

Application of lhrha and hcg to stimulate spermiation and spawning of the toad, Duttaphrynus melanostictus (Schneider, 1799)

In Vietnam, the population of Duttaphrynus melanostictus in the wild has been declined due to overharvesting for snake farms and consumption and environmental pollution. While Black-lipped Toad in cativity rarely ovulates spontaneuosly. Therefore, it’s necessary to study the assisted reproductive techniques to help preserve them. In this study, we used LHRHa and HCG to stimulate induction of spermiation and of spawning of toad. Male were injected with 2 µg/g LHRHa or 3 IU HCG/g to stimulate sperm release. Female were divided in two groups: the first group was administred a single dose of 500 IU HCG (considered as unpriming group) and the second group was administred first two sequential anovulatory dose of 100 IU HCG, repeat after 72 hours. Forty-eight hours after the second injection, the ovulatory dose of 500 IU HCG was injected (priming group). The results showed that injection of LHRHa and HCG stimulates sperm release graetest in time from 5-7 hours after injection. Five hours after injection of LHRHa or HCG, toad sperm concentration was 2.6 and 1.96 million/ml, sperm motility was 82.5 and 85%. Unprimed females failed to spawn any eggs. Whereas, 60% of primed females spawned in the period from 14-16 hours after the last injection of HCG.

Effect of doses and time post-injection on hormonal induction of spermiation in Bufo calamita (Anura: Bufonidae)

Effect of doses and time post-injection on hormonal induction of spermiation in Bufo calamita (Anura: Bufonidae)

The antioxidant system of sterlet seminal fluid in testes and Wolffian ducts

Oxidative stress is a possible source of spermatozoa function deterioration. Seminal fluid (SF) protects spermatozoa against reactive oxygen species (ROS) attack during development in testes and transit through the reproductive tract. Spermatozoa curvilinear velocity and percent of motile cells as well as changes in thiobarbituric acid-reactive substance (TBARS) content, superoxide dismutase, and catalase activity, and uric acid concentration in SF were evaluated in sterlet sperm collected from testes 24 h after hormone induction of spermiation and from Wolffian ducts at 12, 24, 36, and 60 h after hormone injection (HI). While testicular spermatozoa motility was not initiated in activating medium, Wolffian duct sperm showed low motility at 12 h, significant increase at 24 and 36 h, and decrease at 60 h. Testicular SF was characterized by the highest level of TBARS and activity of studied enzymes compared with SF from Wolffian duct sperm at 24 h post-HI. In fluid from Wolffian duct sperm, a significant increase in TBARS content was shown at 36-60 h post-HI. In contrast to testicular SF, in SF from Wolffian duct sperm, this increase was not counterbalanced by changes in the studied variables of antioxidant system. This may be the source of the observed decrease in spermatozoa motility parameters 60 h post-HI. The results may confirm a dual role of ROS in fish sperm physiology. The data with respect to decrease in sturgeon spermatozoa motility parameters at 60 h post-HI should be taken into account in artificial sturgeon propagation.

Buserelin acetate-induced spermiation in Leptodactylus ocellatus and evaluation of semen characteristics

By understanding the hormonal action behind the induction of spermiation in Leptodactylus ocellatus can help in the collection of semen for artificial insemination and cryopreservation. The experiments were conducted at the Experimental Frog Farm of the Federal University of Vicosa. Seven L. ocellatus males with the following secondary sexual characteristics were selected: black thorns, developed forelimbs, and amplexus reflex. Spermiation was induced in these animals by applying daily 0.4 μg buserelin acetate hormone (GnRHa) for 7 days, being the semen collected 90 min after the application. The volume, color, vigor, motility, sperm concentration, and spermatozoa morphology were subsequently evaluated. Three animals responded to GnRHa induction, enabling the collection of seven samples of semen, and the first sample was collected 97.5h after the first application. The semen of the creole frog presented the following characteristics: an average volume of 0.38 mL, murky color, sperm vigor of 3.71, sperm motility of 77.14%, sperm concentration of 6.60 x 10 6 SPTZ mL -1 , and 69% of normal sperm. GnRHa can induce spermiation in the creole frog. Although the volume of collected semen was low, the color, vigor, motility, concentration, and spermatozoa content showed to be adequate.

Sperm characteristics and androgens in Acipenser ruthenus after induction of spermiation by carp pituitary extract or GnRHa implants

Spermiation and changes in androgen (testosterone, T and 11-ketotestosterone, 11-KT) levels were studied in sterlet (Acipenser ruthenus) treated with GnRH agonist implants (DAla(6)-Pro(9)-LHRHa) at 25 and 75 μg kg(-1) b.w. and compared with those males treated with 4 mg kg(-1) b.w. of carp pituitary extract (CPE) and 3 pellets of Ovopel kg(-1) b.w., which contains DAla(6)-Pro(9)NEt-mGnRH and metoclopramide. Sperm quality (sperm mass, spermatozoa concentration and sperm motility and velocity) was evaluated 24, 48 and 72 h after hormonal treatments. Males did not release sperm in the control group injected with physiological solution, while sperm could not be collected 7 days after treatments in all hormonally treated groups. Spermiation rates were 100 % in the CPE and Ovopel groups and 25-50 % in the GnRHa-treated groups. Sperm production was significantly lower in the GnRHa-treated groups than in the CPE and Ovopel groups and decreased 72 h after hormonal treatment. Sperm motility and velocity were higher in the Ovopel and GnRHa (75 μg) groups compared to the CPE and GnRHa (25 μg) groups and decreased 72 h after hormonal treatment. Androgens were only affected in spermiating males and changed in the Ovopel and GnRHa (75 μg) after hormonal treatment. Significant correlations were observed between sperm production, sperm motility and sperm velocity, but not androgens. The present study suggests involvement of dopamine in sturgeon spawning. Additionally, better sperm quality observed in the Ovopel group and particularly sperm motility in the GnRHa (75 μg) suggests enhancement of sperm quality in sturgeon treated with GnRHa. Therefore, further study is needed to induce fully spermiation using GnRHa implants in combination with a dopamine inhibitor.

A comparison of human chorionic gonadotropin and luteinizing hormone releasing hormone on the induction of spermiation and amplexus in the American toad (Anaxyrus americanus)

BackgroundCaptive breeding programs for endangered amphibian species often utilize exogenous hormones for species that are difficult to breed. The purpose of our study was to compare the efficacy of two different hormones at various concentrations on sperm production, quantity and quality over time in order to optimize assisted breeding.MethodsMale American toads (Anaxyrus americanus) were divided into three separate treatment groups, with animals in each group rotated through different concentrations of luteinizing hormone releasing hormone analog (LHRH; 0.1, 1.0, 4.0 and 32 micrograms/toad), human chorionic gonadotropin (hCG; 50, 100, 200, and 300 IU), or the control over 24 hours. We evaluated the number of males that respond by producing spermic urine, the sperm concentration, percent motility, and quality of forward progression. We also evaluated the effects of hCG and LHRH on reproductive behavior as assessed by amplexus. Data were analyzed using the Generalized Estimating Equations incorporating repeated measures over time and including the main effects of treatment and time, and the treatment by time interaction.ResultsThe hormone hCG was significantly more effective at stimulating spermiation in male Anaxyrus americanus than LHRH and showed a dose-dependent response in the number of animals producing sperm. At the most effective hCG dose (300 IU), 100% of the male toads produced sperm, compared to only 35% for the best LHRH dose tested (4.0 micrograms). In addition to having a greater number of responders (P < 0.05), the 300 IU hCG treatment group had a much higher average sperm concentration (P < 0.05) than the treatment group receiving 4.0 micrograms LHRH. In contrast, these two treatments did not result in significant differences in sperm motility or quality of forward progressive motility. However, more males went into amplexus when treated with LHRH vs. hCG (90% vs. 75%) by nine hours post-administration.ConclusionThere is a clear dichotomy between the two hormones’ physiological responses on gamete production and stimulation of amplexus. Understanding how these two hormones influence physiology and reproductive behaviors in amphibians will have direct bearing on establishing similar breeding protocols for endangered species.

Hormonal induction of spermiation, courting behavior and spawning in the southern bell frog, Litoria raniformis

We trialled the efficacy of various exogenous hormones to induce spermiation, courtship behavior, and spawning in the "endangered" southern bell frog, Litoria raniformis. Intralymphatic administration of Lucrin(®), a synthetic nonapeptide luteinizing hormone releasing hormone (LHRH), was used successfully to induce courting behaviors and ejaculation of spermatozoa in males. Various hormones, including Lucrin(®), another synthetic LHRH analog ([des-Gly(10), D-Ala(6)]-LHRH), human chorionic gonadotropin, progesterone, and a dopamine receptor antagonist failed to promote oviposition and spawning in females. This and earlier studies indicate that in the efficacy of hormonal induction in amphibians varies between taxa, hormones, and genders. The lack of response in females may limit the use of reproduction technology in the southern bell frog and closely related species of Australian bell frogs.

Semen characterization and sperm structure of the Amazon tambaqui Colossoma macropomum

Seminal features of tambaqui were evaluated after hormonal induction of spermiation with common carp pituitary extract. Seventeen adult (6.1 ± 0.9 kg, 62 ± 6 cm) males were collected from earthen ponds and transported to indoor concrete tanks. Semen was evaluated according to volume, pH, osmolality, motility, concentration, viability, sperm morphometry and morphological abnormalities. The semen of tambaqui was white and milky. The volume was 10.2 ± 5.1 mL at pH 8.0 ± 0.1, and yielded a concentration of 9.1 × 109 spermatozoa mL−1 while seminal plasma osmolality was 260 ± 7.3 mOsm kg−1. The percentage of viable sperm cells was determined with 97.0 ± 2.0% and 87.0 ± 8.0% using an eosin-nigrosin staining and fluorescent live-dead kit (propidium iodide and SYBR-14), respectively. A negative correlation (−0.65) between semen pH and sperm motility was observed suggesting this feature might influence the tambaqui sperm kinetics. Morphometrically the spermatozoa were on average 35.48 ± 1.55 μm long with a roundish head (mean length: 2.73 ± 0.21 μm; mean width: 2.58 ± 0.18 μm; n = 250 spermatozoa), without an acrosome, and presented a long midpiece (2.90 ± 0.52 μm) and flagellum (29.84 ± 1.63 μm). About 15.8% of the spermatozoa carried morphological abnormalities, with bent tail being the most frequent defect (7.81 ± 3.12%). The characterization of tambaqui semen favors the development of more precise and efficient procedures for its analysis and utilization in controlled breeding.