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Reproductive obstacles have led scientists to develop novel techniques/technologies for artificial reproduction. We aimed to investigate the possibility of propagating zebrafish females using sperm ovarian lavage with and without presence of male stimulus. This experiment consisted of several treatments: traditional spawning approaches with females and wild-type males (AB♀ × AB♂); no males present with non-manipulated females (AB♀); no males present with females inseminated with NaCl into ovarian lobes [AB♀(inj.NaCl)]; no males present with females inseminated with sperm from transgenic males into ovarian lobes [AB♀(inj.Tg♂)]; non-manipulated females kept separately from wild-type males (AB♀|AB♂); females kept separately from wild-type males and inseminated with NaCl into ovarian lobes [AB♀(inj.NaCl)|AB♂]; and females kept separately from wild-type males and inseminated with sperm from transgenic males into ovarian lobes [AB♀(inj.Tg♂)|AB♂]. There were no released eggs in both negative control treatments (AB♀ and AB♀|AB♂). Egg production increased (ranged from 0 to 28.5 eggs/female) when females were injected in the presence [AB♀ (inj.NaCl) |AB♂] or absence of male stimulus [AB♀ (inj.NaCl) and (AB♀(inj.Tg♂)]. A further increase in egg production [relative to AB♀, AB♀ (inj.NaCl), and AB♀|AB♂] was detected when females were inseminated with pooled sperm from transgenic males in the presence of male stimulus [AB♀(inj.Tg♂)|AB♂; ranged from 2.5 to 55 eggs/female] or when using traditional spawning approaches (AB♀ × AB♂; ranged from 25 to 131 eggs/female). Females inseminated with sperm produced embryos, although no differences were detected when females were inseminated with pooled sperm from transgenic males in presence (11.8 ± 16.3%) or absence (average = 12.6 ± 9.2%) of male stimulus. Traditional spawning approaches produced the most eggs (81.2 ± 42.3 per female) and highest fertilization rate (81.3 ± 10.4).

Aquaculture of red swamp crayfish, Procambarus clarkii (Girard, 1852), has developed rapidly worldwide in recent years with promising prospects. However, limited knowledge about temperature effects on reproductive performance and embryonic development has hindered the development of crayfish aquaculture. The two present studies were conducted to identify optimal water temperatures (17 °C, 21 °C, 25 °C, 29 °C and 33 °C) for reproductive performance (experiment 1) and embryonic development (experiment 2) of P. clarkii. Totally, there were 12 replicates, with 480 adults and embryos from 60 ovigerous crayfish selected for experiment 1 and 2, respectively. In the first experiment, the survival of adult crayfish was not significantly affected by the temperatures tested. However, significantly higher feeding rates, spawning rates, and fecundity were obtained at 21 °C and 25 °C when compared to those at 29 °C and 33 °C. Polynomial models and loess regression fitted to the experimental data showed that highest spawning rates and fecundity occurred at 21 °C while shortest duration from mating to spawning was found at 33 °C. In the second experiment, we found that optimal embryonic development was at 25 °C with shorter hatching time and no abnormalities observed. However, while embryos showed abnormalities and subsequently died at 29 °C and 33 °C. We further built a temperature-dependent developmental model for P. clarkii embryos: D (developmental time, days) = 3,140,837(T-2.03)−3.76. Based on these results, the temperature range 21 °C – 25 °C was recommended for adult crayfish reproduction and 25 °C was recommended for embryonic development. This study indicates that manipulating water temperature is an effective alternative to current artificial reproduction techniques (e.g. eyestalk ablation and injection hormones) to induce spawning and embryonic development and thus provides mass production of juvenile P. clarkii for aquaculture.

Goat production is central to resource-poor rural livelihoods in the tropics. However, it is constrained by high inbreeding levels and poor access to good quality breeding bucks. In this study, semen extenders differing in egg yolk and energy level were compared in their ability to maintain liquid semen from a tropical goat breed, when stored at physiological (37 °C) or refrigerator (4 °C) temperature. Semen was collected weekly for nine weeks from seven bucks of the Small East African goat breed, diluted in three extenders and incubated at 37 °C or 4 °C for 24 h. The semen parameters: viability, motility and morphology were better preserved when semen was incubated at 4 °C compared to 37 °C across all extenders. Sperm motility after 24 h at 4 °C was 50.4 ± 5.5%, 50.4 ± 6.2% and 54.3 ± 5.4% for Extender 1 (high glucose, 18% egg yolk), Extender 2 (low glucose, 2.5% egg yolk) and Extender 3 (low fructose, no egg yolk), respectively. Extender 2 maintained significantly higher viability values (67.7 ± 2.5%) at 24 h compared to Extenders 3 (59.3 ± 2.1%) and 1 (46.7 ± 4.2%). Normal morphology after 24 h (88.1 ± 3.1% at 4 °C and 85.6 ± 3.1% at 37 °C) fell within the acceptable range for good quality semen. Acceptable quality was maintained within 8 h and up to 24 h from collection for non-frozen goat semen stored at 37 °C and 4 °C temperature, respectively. These results imply that Small East African goat semen diluted in low or non-egg yolk based extenders and stored at refrigerator temperature can be valuable for artificial reproduction of native goats on low resource capacity farms in developing countries.

[EN] Maturation in captivity of European eel (Anguilla anguilla) requires long and costly hormonal treatments that often lead to asynchronic maturation between sexes. Therefore, optimization of sperm short-term storage methods and cryopreservation protocols can be a key factor for successful artificial fertilization. Two experiments were carried out to optimize the existing protocols. For the short-term storage experiment, sperm was diluted in P1 extender and then stored at different dilution ratios (1,9 and 1,49). The best outcome was then tested at different temperatures (4 and 20¿°C) and in constant agitation or still. In the cryopreservation experiments, large sperm volumes (cryotubes of 2 and 5¿ml), different cooling rates (freezing tubes 1 or 3¿cm above liquid nitrogen during 15 and 20¿min), and different extender compositions (methanol 10% was used as cryoprotectant, and complemented with FBS 20%, BSA 5% or egg yolk 5%) were tested. Sperm kinetic parameters were analyzed with a CASA-Mot system both in fresh and short- or long-term stored samples. In the short-term storage trial, sperm quality did not show significant differences in the first 24¿h after sperm collection between the different storage conditions tested. For longer time, 1:49 dilution ratio showed significantly better results than 1:9, and low temperature (4¿°C) was better for sperm preservation after 3¿days. Cryopreserved sperm samples showed good motility results when they were frozen in cryotubes of 2 and 5¿ml, with no significant differences compared to samples cryopreserved in lower volumes (straws of 0.5¿mL). Furthermore, the combination of methanol (10%) and egg yolk (5%) as freezing medium, induced significant higher post-thawing motility values (over 50%) than the control (methanol 10%), whereas the addition of FBS (20%) and BSA (5%) led to a significant reduction of the sperm motility. The establishment of these storage and cryopreservation protocols will be important for the improvement of European eel artificial reproduction programs., Funded by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement N° 642893 (IMPRESS), including the JGHJ and CR predoctoral contracts. VG has a postdoc grant from the UPV (PAID-10-16).

Persian sturgeon (Acipenser persicus), a commercially valuable and critically endangered fish species has been suffering considerable declines in populations in the nature due to over-fishing, habitat destruction and marine pollution during past decades. Since there were no achievements in artificial reproduction programs, genetic resource banking such as gametes and embryo cryopreservation can be a good strategy however, reported resulting gamete qualities were considerably low. In the present study, the metabolome content of Persian sturgeon spermatozoa was investigated during common straw cryopreservation and novel droplet vitrification by the use of 1H NMR (Nuclear Magnetic Resonance) spectroscopy. Univariate (ANOVA) and multivariate (PCA) analysis showed significant differences in the metabolic profiles between cryopreserved and fresh spermatozoa samples. Adenine, creatine, creatine phosphate, glucose, guanidoacetate, lactate, N, N-dimethylglycine, and glycine levels showed no significant differences between these two cryopreservation techniques suggesting these metabolites and their corresponding enzymes and chemical pathways are so vulnerable to the temperature changes and even higher cooling rate in droplet vitrification could not conserve them. However, significant differences were found in acetate, creatinine, betaine, β-alanine and trimethylamine N-oxide suggesting better efficiency of droplet vitrification in protection of some metabolites associated to spermatozoa energetics, redox balance and hypoxia compensation compared to straw cryopreservation.

Pinctada martensii is one of several important pearl oysters. Ammonia is typically added to the seawater and used during artificial reproduction to improve the fertilization rate. However, the optimal ammonia concentration has not been determined. To determine the optimal ammonia concentration for fertilization success, the effect of the ammonia concentration on the fertilization rate in P. martensii was investigated under the simultaneous influence of temperature and salinity. A Box-Behnken design and response surface method were used in this study. The results showed that the ammonia concentration had a significant effect, which varied quadratically with the ammonia concentration in the ranges of the factor regimes (P

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.

The aim in the present study was to evaluate the effects of motile sperm:oocyte ratio and the use of thawed spermatozoa at different post-activation times in artificial reproduction of gray catfish (Rhamdia quelen). Cryopreserved sperm samples were evaluated for sperm motility and velocity using Computer Assisted Sperm Analysis (CASA). The sperm activation curves were generated using a non-linear statistical model and were used to assess the spermatozoa velocity after thawing. Thus, the oocytes were mixed with thawed sperm at a motile sperm:oocyte ratio of 70,000, 90,000, 110,000, 130,000, 150,000 and 170,000. The thawed sperm were used at 11, 16 and 30 s after spermatozoa activation. At these times, the sperm velocities corresponded to 52, 37 and 21 μm/s. The effects of experimental factors (spermatozoa:oocyte ratio and time after sperm activation) on oocyte fertilization, egg hatching, and percentage of normal larvae were evaluated. The response surface analysis indicated there was no interaction (P 0.05) between the motile spermatozoa:oocyte ratio and time after sperm activation on fertilization, hatching or percentage of normal larvae. The time after sperm activation, however, affected (P 0.05) in a directly proportional waythe oocyte fertilization and egg hatching rates. The time after sperm activation affected the sperm velocity and oocyte fertilization and egg hatching rates. Thus, the use of thawed sperm immediately after sperm activation or with the greatest sperm velocities (11s; 52 μm/s; 62.59% motility) at a relatively lesser motile sperm:oocyte ratio (70,000:1) allows for acceptable fertilization (48.68% for fertilization; 29.61% for hatching) in Rhamdia quelen.

The present study aimed at evaluating the effects of different thermal regimes and synthetic hormones on sperm quality as well two (low vs. high) salmon gonadoliberin (sGnRHa) dosages on egg quality in indoor-reared pikeperch. Two groups of females were formed receiving either 5 µg kg−1 or 50 µg kg−1 of sGnRHa. Likewise, four groups of males were formed that were hormonally treated at either 5 °C or 9 °C, and each temperature receiving either 25 µg kg−1 of sGnRHa or 10 µg kg−1 recombinant human chorionic gonadotropin (hCGrec). Each group consisted of five specimens. Low dosage of sGnRHa showed lower survival of embryos (17.4 ± 29.1% vs. 50.3 ± 7.3%), while there were no differences in sperm quality parameters among the male groups. Overall, best male performance was seen in the group injected with sGnRHa at 9 °C, having the greatest values for all but one of the evaluated parameters. A higher dosage of 50 µg kg−1 can be supported as optimal for a stable thermal regime in pikeperch artificial reproduction, while the differences in the physiological state of breeders under the different thermal regimes should be of interest in future studies. No differences in assessed sperm quality parameters indicate that males are tolerant to thermal manipulations while synthetic form of gonadotropin is applicable for pikeperch propagation and should be evaluated in a more comprehensive study.

The burbot (Lota L.) is one of the most promising freshwater new fish species for cold-water aquaculture. Due to the lack of availability of wild spawners, it is imperative to breed cultured spawners. In the present study, artificial reproduction of a cultured stock of burbot was carried out and then compared with wild fish reproduction results. Patterns of burbot final oocyte maturation were developed and they were compared during artificial reproduction of wild and cultured burbot. For the first time, a model of final oocyte maturation (FOM) was formulated in a hormonally stimulated female burbot. The relationship between the oocyte maturity stage is straightforward and, at a water temperature of 2 °C, progress by the next stage was observed in two-day intervals. This model was tested on a group of wild and cultured fish (generation F1) originating from the Vistula Lagoon (Northern Poland). In both cases, the very high utility of the proposed model in the artificial reproduction of burbot was confirmed. The fertilization was very high. In wild and cultured forms was similar and was over 95% (95.4 ± 0.3 and 95.3 ± 0.3 as mean ± SD for wild and cultured, respectively). The differences in the survival to eyed-egg-stage (92.2 ± 0.8 and 91.9 ± 0.7 for wild and cultured, respectively) and hatchability of eggs (91.1 ± 0.9 and 90.9 ± 0.8 for wild and cultured, respectively) were statistically insignificant. In summary, a cultured stock of burbot spawners can be successfully used for the stable production of the stocking material of this species.

Patagonian blenny (E. maclovinus) is a marine species recently placed in captivity and which are potentially farmable. Understanding and improving its sperm capacity to withstand short-term storage conditions is a key element of initiating an artificial propagation program for this species. The aim of this study is to evaluate the ultrastructure and quality of E. maclovinus sperm during refrigerated storage. To address this objective, scanning electron microscopy (SEM), cytofluorimetric analysis (membrane integrity; reactive oxygen species generation; mitochondrial membrane potential) and cell respiration/mitochondrial-function analysis (ATP content; oxygen consumption) could be useful for optimizing or improving management for artificial reproduction of this species. Severe damage of plasma membranes was observed by SEM at day 7 and 14 of in vitro storage. Analyses of sperm quality were conducted during the 14-day cold storage period when sperm were in diluted (with Cortland solution) and undiluted conditions. When there were diluted conditions, there was greater preservation of motile capacity (from day-7; P 0.05), membrane integrity (from day-7; P 0.05), mitochondrial membrane potential (from day-10; P 0.05) and ATP stores (from day-3; P 0.05). Oxygen consumption indicators were 18.6% ±14.7% greater in the undiluted samples from day-3, and 32.1%±2.1% of the total spermatozoa had ample amounts of superoxide anion in both undiluted and diluted semen on day-0. The use of Cortland solution extended the viability of sperm when there were longer storage times. Factors that have a greater effect on the quality of semen during storage are reactive oxygen species generation and ATP depletion. In conclusion, Patagonian blenny spermatozoa can be stored at 4 °C between 7 and 10 days using Cortland solution.

Understanding the effects of environmental factors on sperm motility characteristics can increase artificial reproduction efficiency in species that do not spawn naturally in captivity, such as Megaleporinus obtusidens. This study evaluated the effects of the osmolality (25, 85, 145, 205, 265, and 325 mOsm kg−1) and composition of activating solutions (NaCl, KCl, or fructose) on the percentage of motile sperm, and the swimming velocity and path straightness of M. obtusidens spermatozoa. The concentrations of major ions in the seminal fluid were also assessed and Na+ (74.46 mmol L−1), K+ (37.24 mmol L−1), and Cl− (114.29 mmol L−1) were the most abundant. When the activating solution was hypertonic (325 mOsm kg−1) compared to the seminal fluid (293 mOsm kg−1), sperm motility was completely inhibited. A wide range of osmolalities that initiated sperm motility were identified for all three solutions. Both, the percentage of motile sperm and the motility duration were reduced (P 50%) up to 40 s after activation and the motile phase lasted for >50 s, regardless of the activating solution composition. Over the postactivation time, the curvilinear velocity and straightness were similar (P > .05) for fructose and NaCl solutions, whereas KCl solutions induced a higher (P

In this study the comparison of spontaneous and hormone-induced reproduction of burbot under controlled conditions was made. During the first two years of the study, burbot spawned spontaneously and artificially after hormonal treatment. The application of sGnRHa resulted in a 100% ovulation rate within 4 to 5 days. Over the 20 days of the experiment, in both years, 65% of females (13 from 20) spawned spontaneously. The biological quality of eggs, expressed as a percentage of live embryos (58.6–60.3%), was generally lower than in only hormone-induced spawned groups (84.2–89.2%). The embryo survival in groups spawned spontaneously decreased in time during the spawning season. Hormonal stimulation of non-ovulated females caused 43–57% of ovulation but with low embryo survival rates (34.2–44.2%). In the third year of the study, 55% of females ovulated spontaneously during the first 10 days of the experiment. Stimulation of non-ovulated females resulted in final oocyte maturation and ovulation in 78% of females. The embryo survival rate was higher (63.2%) than in fish stimulated hormonally after 20 days of spontaneous spawning (but not after 10 days). The application of hormonal treatment in burbot reproduction under controlled conditions shortened latency time, synchronized final oocyte maturation and resulted in high embryo survival rate.

Reproduction of amphibians includes ovulation, spermiation, fertilization, oviposition, larval stage and development, and metamorphosis. A problem at any stage could lead to reproductive failure. To stimulate reproduction, environmental conditions must be arranged to simulate changes in natural habits. Reproductive life history is well documented in amphibians; a thorough knowledge of this subject will aid the practitioner in diagnosis and treatment. Technologies for artificial reproduction are developing rapidly, and some protocols may be transferable to privately kept or endangered species. Reproductive tract disorders are rarely described; no bacterial or viral diseases are known that specifically target the amphibian reproductive system.

Anesthesia is a common practice used in fish research and aquaculture. It is important to understand anesthetic effects on the animal and tissues of interest to ensure validity of data and to improve animal welfare in research and fish production endeavors. The production of some captive fish species is only possible by imposing artificial reproduction procedures, and manipulation of fish for these purposes is a stressor. The purpose of this study, therefore, was to evaluate effects of different concentrations (100, 200, and 300 mg/L) of the anesthetic MS-222 (tricaine methanesulfonate) on cortisol concentrations and effects on sperm quality in Rhamdia quelen. After hormonal induction of gamete production, 28 sexually mature males were randomly assigned to treatments, and milt and blood samples were collected. Anesthesia induction time, motility rate, sperm concentration and morphology, plasma cortisol concentrations, and reproductive hormone concentrations (testosterone, 17-α-hydroxyprogesterone, and estradiol) were evaluated. Sperm motility was greater in the control than 300 mg/L treatment group but did not differ among the control, 100, and 200 mg/L groups. The estradiol concentration was greater in non- anesthetized than anesthetized Rhamdia quelen, but plasma cortisol concentrations did not differ among treatment groups (182.50 ± 42.03 ng/mL). The anesthetic MS-222 at concentrations of 100, 200, and 300 mg/L did not inhibit the stress response due to handling of Rhamdia quelen males. In addition, treatment with MS-222 was not effective in inhibiting detrimental effects on sperm quality because this treatment was associated with impaired sperm motility and lesser concentrations of plasma estradiol.

The aims of this study were to investigate (a) the effects of single or multiple sperm collection with catheter, and (b) impacts of multiple sampling method (catheter or syringe) on sperm quality in pikeperch (Sander lucioperca). To compare the effects of sampling method, sperm was collected repeatedly from the same fish using catheter (n = 6) or syringe (n = 5) at 48, 52 and 56 h post-hormonal injection. For the single collection group as control, sperm was collected with catheter from 12 fish at 48 h post-injection. The results showed that multiple sperm collection with catheter does not significantly (P ˃ 0.05) influence sperm volume and density compared to single sperm collection. However, multiple collection with syringe led to drastic reduction of sperm density at 56 h post-injection. Moreover, sperm volume was influenced by post-injection time where the highest volume was obtained at 48 h post-injection. Furthermore, sperm density and osmolality of seminal plasma were significantly affected by sampling method and post-injection time where higher values were found for the samples collected with catheter at 48 h post-injection. Number of males producing milt with motile spermatozoa decreased along with multiple sperm collection. Sperm motility at 60 s post-activation was affected by the interaction of sampling method and post-injection time, and the highest value was obtained for the sperm collected with syringe at 48 h post-injection. Sperm curvilinear velocity (VCL) at 15 and 45 s post-activation was affected by sampling method and post-injection time, respectively. These findings contribute to the pikeperch hatchery practices, as multiple sperm collection could be applied during artificial reproduction if limited number of males are available.

African weakly electric mormyrid fish show a high diversity of their electric organ discharge (EOD) both across and within genera. Thanks to a recently developed technique of artificial reproduction in mormyrid fish, we were able to perform hybridizations between different genera and within one genus (Campylomormyrus). The hybrids of intergenus hybridizations exhibited different degrees of reduced survival related to the phylogenetic distance of the parent species: hybrids of the crosses between C. rhynchophorus and its sister genus Gnathonemus survived and developed normally. Hybrids between C. rhynchophorus and a Mormyrus species (a more basal clade compared to Campylomormyrus s) survived up to 42days and developed many malformations, e.g., at the level of the unpaired fins. Hybrids between C. numenius and Hippopotamyrus pictus (a derived clade, only distantly related to Campylomormyrus) only survived for two days during embryological development. Eight different hybrid combinations among five Campylomormyrus species (C. tamandua, C. compressirostris, C. tshokwe, C. rhynchophorus, C. numenius) were performed. The aim of the hybridizations was to combine species with (1) either caudal or rostral position of the main stalk innervating the electrocytes in the electric organ and (2) short, median or long duration of their EOD. The hybrids, though they are still juveniles, show very interesting features concerning electrocyte geometry as well as EOD form and duration: the caudal position of the stalk is prevailing over the rostral position, and the penetration of the stalk is dominant over the non-penetrating feature (in the Campylomormyrus hybrids); in the hybrid between C. rhynchophorus and Gnathonemus petersii it is the opposite. When crossing species with long and short EODs, it is always the long duration EOD that is expressed in the hybrids. The F1-Hybrids of the cross C. tamandua×C. compressirostris are fertile: viable F2-fish could be obtained with artificial reproduction.

Reducing egg stickiness is a crucial step in artificial culture of sturgeon eggs, with commonly used methods being time consuming or interfering with hatching. Sodium hypochlorite (SH) at varying concentrations and exposure times was tested on sterlet Acipenser ruthenus eggs, with 0.03% SH for 40 s effectively eliminating stickiness without impairment to eggs, embryo development, or hatching when compared with other de-adhesion methods (clay; NaCl, urea, and tannic acid). Field tests using sterlet, Siberian sturgeon Acipenser baerii, and Russian sturgeon Acipenser gueldenstaedtii showed similar numbers of larvae hatched using SH and clay. Immunohistochemistry revealed a protein carbonyl group only on the surface of eggs treated with SH, evidence of oxidation as the mode of action and indicating that oxidation did not affect inner egg layers or cytoplasm. Treatment with SH is a rapid, simple, and inexpensive method for de-adhesion of sturgeon eggs. Statement of relevance Sturgeons represent a valuable group of fish in aquaculture, especially for caviar, but also for meat production. This study describes a novel approach to sturgeon egg de-adhesion involving oxidation of egg surface. The method requires little time, is low cost, and disinfects and softens the egg surface, which facilitates hatching.

To identify the effects of oocyte ageing on egg quality in northern pike, Esox lucius , two experiments were performed as follows: in experiment I, partial volumes of the eggs from 7 pike females were stripped and fertilised with 2-day intervals for 8 days post-ovulation (DPO). In experiment II, the stripped eggs of 3 females were stored in ovarian fluid at 10 °C and fertilised separately with 12-hour intervals for 96 hours post-stripping (HPS). In both experiments, the eggs from each batch were fertilised with mixed milt obtained from 5 males. Eyeing, hatching and eyed-egg mortality rates as well as the occurrence of larval malformations and ploidy anomalies were considered as indices for egg quality. The results of experiment I indicated that the best and most reliable post-ovulatory stripping times in pike were at 2 DPO and up to 4 DPO, respectively, when females were retained at 10 °C. Over-ripening of the eggs occurred from 8 days after ovulation. The eyed-egg mortality and larval malformation rates did not show any significant increase up to 6 DPO, but then increased significantly and reached 36% and 43% at 8 DPO, respectively. The incidence of triploid larvae increased over time, from 0% at 0 and 2 DPO to 11% and 14% in more aged oocytes at 4 and 6 DPO, respectively. The second experiment clarified that the best post-stripping fertilisation time in pike was at 0 HPS. However, stored eggs in ovarian fluid could retain almost 50% of their initial fertilising ability for 24 h after stripping. Complete loss of egg viability occurred after 96 HPS, and the highest eyed-egg mortality rate (27%) and larval malformation rate (33%) were detected in this group. The obtained results suggest that for commercial artificial reproduction procedures in northern pike, the time interval between successive fish examinations for ovulation should not exceed 4 days. In addition, the eggs 1 stripped from the female should be fertilised immediately following collection to secure the highest possible fertilisation rate. Statement of relevance Knowledge of in vivo and in vitro storage of fish eggs can improve hatchery techniques as well as better broodstock management, which are the most important factors affecting synchronization of the artificial fertilisation and consequently the effectiveness of the entire operation. The duration of egg viability after ovulation and stripping has not yet been determined for the pike. A better understanding and refinement of egg storage methods are strongly desirable to maximize the efficacy of mass production in pike hatcheries.

Eyeing and hatching rates with regard to sperm: egg ratio, the length of time eggs remain viable post-activation in water, and duration of storage time in testicular and stripped sperm were determined in northern pike Esox lucius. In a trial of seven sperm: egg ratios, we found 4 × 105 to be the minimum tested number of spermatozoa per egg to produce an acceptable hatching rate (66 ± 4%). To determine the period of time that eggs remain viable after activation, eggs were placed in hatchery water and fertilized at 0, 15, 30, 45, 60, 90, 120, 240, and 480 s post-activation. With elapsed time, the eyeing and hatching rates decreased significantly (P

The present study aimed at the development of an effective and efficient artificial reproduction technique for cultured female tongue sole (Cynoglossus semilaevis) in out-of-season. After about four-month enriching cultivation of broodstocks, a series of experiments were conducted to develop a protocol for the hormonal induction of ovulation and artificial fertilization. The results showed that single injection of combination of luteinizing hormone releasing hormone analogue (LHRH-A3), human chorionic gonadotropin (HCG) and domperidone (DOM) is effective to induce ovulation at different final maturation stages (i.e., stage IV and V). The optimal single injection dosages of combined hormones were (0.15 μg LHRH-A3 + 4 IU HCG + 2 mg DOM) / kg BW and (0.05 μg LHRH-A3 + 2 IU HCG + 1 mg DOM) / kg BW for females at stage IV and V respectively. At the optimized dose of hormones, the fertilization and hatching rates were 44.6% and 80.4%, 30.4% and 63.4% for stage IV and V respectively. The lapsed time to ovulation was very predictable and ranged from 35.5 to 39.5 h post injection. Ovulated eggs in ovarian cavity should be stripped within 15 min in order to obtain the highest fertilization and hatching rates, otherwise the values decreased significantly thereafter. These researches are expected to provide improved implementation of artificial reproduction techniques for the production of fertilized eggs in the commercial aquaculture of tongue sole.

When artificial reproduction technologies designed for use with horses are used with donkeys, success is dependent on awareness of the physiological differences between these species, yet little information is available on many aspects of donkey reproduction. The present work examines the activity of the CL in Catalonian jennies after induced luteolysis. Plasma progesterone concentration, luteal blood flow (determined by color Doppler), and CL cross-sectional area (CL-CSA; determined by B-mode ultrasound examination) were assessed after a single dose (5 mg intramuscular) of dinoprost thromethamine (DT, a PGF2α analog) on Day 10 after ovulation in two experiments. In experiment 1, a preliminary experiment, data were collected daily for 4 days after DT administration. Values for all the measured variables decreased over this period. In experiment 2, data were collected during the first 24 hours after DT administration because in experiment 1, most luteolytic activity occurred during this time. An increase in luteal blood flow was seen between 0 and 3 hours, followed by a progressive reduction, whereas the values for plasma progesterone and CL-CSA gradually decreased from 0 hours onward. In both studies, negative correlations were seen between all variables and the time of sampling. In contrast, positive correlations were seen between plasma progesterone, CL-CSA, uterine tone, and luteal blood flow. Indeed, a strong correlation was recorded between plasma progesterone and luteal blood flow (r = 0.70; P

There is very little scientific literature about the impact of Eel virus European X (EVEX) on eels held under artificial reproduction condition, and its effective role in eel decline is uncertain and still unclear. Following a disease outbreak of European eels, farmed in an experimental hatchery in Italy, a sample of the affected animals was analyzed to determine the cause of illness. Virological isolation, PCR assay and molecular characterization revealed the presence of EVEX in the diseased animals. The complete N, P, M and G gene regions were amplified by using novel primers specific for EVEX. The EVEX strain identified in this study was phylogenetically close to the known EVEX isolates; however, this Italian isolate diverges and localizes in a separated branch of this cluster. In addition, some unique amino acid changes in three out of four analyzed viral proteins differentiated the Italian virus from reported EVEX sequences which, although few in number, do cover different geographical areas and a 30-year time span. Molecular data are needed to gain further insight into the genetic variability of EVEX, to better define its phylogenetic and evolutionary relationships and to unravel the existence of different circulating strains. Examination of samples caught in the same area failed to detect the presence of EVEX infection. These results strongly indicate that the presence and risk factors associated with EVEX infection are not well established and further study should be performed in this field, in particular on stress and temperature dependence for disease development.

Based on the reports of unsuccessful ovulation in pacu (Piaractus mesopotamicus) by fish farmers and researchers undertaking artificial reproduction programs, we evaluated the use of prostaglandin F (PGF) to improve pacu ovulation. This study was conducted during two spawning seasons (2009/2010 and 2010/2011) with two samplings in the first season and one sampling in the second season. A total of 45 females was sampled in this study. The control group was injected with carp pituitary extract (crude extract, 6 mg/kg), and the treatment group received PGF (2 mL per fish in the 2009/2010 season and 5 mL per fish in the 2010/2011 season) in addition to the crude extract. In both seasons, 100% (N = 4, 2009/2010 first sampling; N = 5, 2009/2010 second sampling; and N = 3, 2010/2011) of the PGF-treated fish spawned. In contrast, 53.0% (N = 9) and 83.3% (N = 10) of the control fish spawned in the first and second samplings of the 2009/2010 season, respectively, and only 25.0% (N = 1) spawned in the 2010/2011 season. Fecundity, fertility, and hatching rates did not differ (P > 0.05) between the treated and control fish. Based on oocyte volume frequency analysis, ovaries of the control fish had more (P < 0.05) vitellogenic oocytes with germinal vesicle breakdown that remained unovulated after spawning, whereas more (P < 0.05) of previtellogenic oocytes were present in the ovaries of the PGF-treated fish. In conclusion, administration of exogenous prostaglandin may improve the outcome of hormonally induced spawning in tropical migratory fish.

Most fishes with commercial importance from the São Francisco basin are migratory and do not complete the reproductive cycle in lentic environments, such as hydroelectric plant reservoirs, hence natural stocks are declining and there is an urgent need to reduce the pressure of fishing on those wild populations. Therefore, studies on reproductive biology and its relationship with endocrine and environmental factors are key to improving the cultivation techniques of Brazilian fish species. This study examined the influence of water temperature on sex steroid concentrations (testosterone, 17β-estradiol and 17α-hydroxyprogesterone), spawning efficiency, fecundity, fertilisation rate, larval abnormality rates and involvement of the cytoskeleton during the final oocyte maturation of Prochilodus argenteus under experimental conditions. The results of our study showed that in captivity, sex steroid plasma concentrations and spawning performance of P. argenteus were clearly different for fish kept in water with different temperature regimes. In lower water temperature (23°C), it was observed that: 33% of females did not ovulate, fecundity was lower and vitellogenic oocytes after the spawning induction procedure exhibited a smaller diameter. Moreover, concentrations of 17β-estradiol and 17α-hydroxyprogesterone were lower and there was a delay in the final oocyte maturation and, consequently, ovulation and spawning. Our experiments showed direct influence of water temperature in the process of induced spawning of P. argenteus. Changes in water temperature also suggest the tubulin involvement in the nuclear dislocation process and the possible action of actin filaments in the release of polar bodies during final oocyte maturation of P. argenteus.

Artificial reproduction of asp under controlled conditions was investigated using two different spawning agents based on luteinizing hormone releasing hormone (LHRH) analogues and dopamine antagonists (Ovopel and Ovaprim), during two successive spawning seasons. Fish in the Ovopel and Ovaprim treatment groups were treated with a dose equivalent to 2 pellets and 0.5 mL per kg of body weight respectively. Out-of-season spawning and reproduction after a sudden increase in water temperature were also examined. No control fish ovulated although in both years, fish treated with Ovaprim showed a higher percentage of ovulation (75–88%) and embryo survival to the eyed-egg-stage (69–78%) than those treated with Ovopel (ovulation 25–38%; embryo survival 36–47%). The latency time was shorter in the groups where Ovopel was applied (40–42 h) than in Ovaprim groups (42–48 h). Similar results were obtained in out-off-season spawning. After a sudden increase in water temperature, females treated with Ovopel did not ovulate. This indicates that Ovaprim might be successfully used for artificial reproduction of asp by applying the induction protocol and the conditions reported in the present study.

Egg incubation is critical for artificial reproduction of turtles, but how incubation conditions affect embryos and resultant hatchlings remains unknown in many species. We investigated the influence of temperatures on incubation period, hatching success and hatchling traits to indentify the optimal temperatures for egg incubation in two Geoemydid turtles ( Ocadia sinensis and Mauremys mutica ). Incubation period of O. sinensis eggs was shorter than that of M. mutica eggs. In both species, hatching success was high in the temperature range of 26–30 °C, and hatchlings were mainly males if incubated at low temperatures and females at high temperatures. The pivotal temperature for sex determination fell into the range of 28–30 °C for O. sinensis and 26–28 °C for M. mutica . Incubation temperature profoundly affected post-hatching growth in M. mutica , but not in O. sinensis . Hatchling M. mutica from 24 and 26 °C grew faster than those from 28 and 30 °C. Therefore, temperatures around 26 or 28 °C would be optimal incubation temperatures for M. mutica or O. sinensis to improve hatching success and post-hatching growth, and to yield offspring with a balanced sex ratio. Moreover, this study demonstrates significant inter- and intra-specific variations in optimal incubation temperature and pivotal temperature, which implies that simply adopting incubation regimes developed for other species or populations should be avoided in turtle aquaculture.

The pirapitinga (Piaractus brachypomus) is a freshwater fish that inhabits the Amazon and Orinoco River basins. The use of cryopreserved sperm has been considered to facilitate procedures during the artificial reproduction. The aim of the present study was to develop a freezing protocol for pirapitinga sperm collected outside the spawning season. Sperm samples were diluted in four freezing media prepared by a combination of two extenders (glucose and BTS-Beltsville Thawing Solution) and two cryoprotectant agents (DMSO and methylglycol) loaded into 0.5-mL straws, frozen in a nitrogen-vapor shipping dewar (dry-shipper) and stored in liquid nitrogen at -196 degrees C. Post-thaw sperm motility was evaluated both subjectively using a light microscope and by a computer-assisted sperm analyzer (CASA). Curvilinear, average path and straight-line velocities were also determined. There were no differences (P>0.05) in post-thaw sperm motility between evaluations performed subjectively and using the CASA. Sperm samples cryopreserved in glucose-methylglycol yielded the greatest post-thaw sperm motility (81%) and fastest sperm velocities when compared to the samples frozen in the other three media (P

Efficiency of Ovopel (mGnRHa + metaclopramide) and Ovaprim (sGnRHa + domperidone) treatment on the quantity and quality of European smelt milt were tested. Either preparation resulted in stimulation of smelt sperm production (100% spermiating males vs. 68% in control). At the dosage tested, Ovaprim treatment increased the volume of milt and total sperm production, whereas Ovopel treatment produced sperm with a higher percentage of motility. Among computer-assisted sperm motility analysis (CASA) parameters only amplitude of lateral head displacement (ALH) significantly differed from control in both hormonal treated groups. For most parameters no significant differences between control and stimulated males were found. Our data suggested that administration of two different GnRHa combined with two different dopamine antagonists would help in the future work for improving procedures of artificial reproduction and conservation of European smelt.

The DNA damage in human spermatozoa is a relevant predictor of prognosis in male infertility, whereby increased sperm DNA damage impairs the outcomes of artificial reproduction. Theoretically, DNA damage should alter the special cellular functions of human spermatozoa, and lead to diminished acrosome reaction with reduced fertilization rates. Nevertheless, intracytoplasmic sperm injection (ICSI) has been reported to alleviate such negative outcomes due to DNA damage. This study investigated the relationship between DNA fragmentation and acrosome reaction as well as viability in ICSI patients. The study enrolled 42 men undergoing ICSI due to poor sperm parameters. The DNA fragmentation indexes (DFI) were 4-10% in 38% of the cases, andor = 10% in 19% of the cases. The results of both acrosome reaction and viability assays showed negative correlations with DFI values in all cases and especially in cases with fertilization rates60% (P0.05). However, such correlations were not found in cases with fertilization rates60%. There were no live deliveries in patients with high DFI levels (10%). In conclusion, negative correlations were identified between increased DNA damage, and acrosome reaction and/or viability of human spermatozoa, especially in cases with reduced fertilization rates.

Methods and results of recent hatchery studies in rearing the clam, Xishi tongue, Coelomactra antiquata (Spengler) are documented in this paper. About 17 million juveniles were produced using hatchery methods, the largest production ever reported. A summary of its biological and ecological characteristics and techniques for hatchery rearing and growout are reported. Because of good growth rate, ease of artificial breeding and growout, this famous Chinese delicacy is a promising new candidate for aquaculture. This indigenous Chinese species has excellent potential for aquaculture; and due to its decline in natural abundance, hatchery rearing and growout may help the conservation of this species in the wild.

The effects of temperature and salinity on eggs and larvae of the leopard grouper, Mycteroperca rosacea, were studied. Eggs from hormone-induced spawning were artificially fertilized. The effect of temperature (20, 24, 26, 28, and 30 °C) on hatch rate, notochord length, yolk sac volume, and oil droplet diameter was recorded. Times from fertilization to 50% hatch and hatching period duration (first to last hatch) were also recorded for each temperature. The effect of temperature (20, 24, 26, and 28 °C) on survival, growth rate, and final larval lengths 5 days after fertilization was demonstrated. The effect of salinity (0‰, 8‰, 16‰, 24‰, 32‰, 40‰, 48‰, 56‰, and 64‰) on hatching rate and larval lengths at hatch was recorded. Additionally, the effect of salinity (0‰, 10‰, 20‰, 30‰, and 40‰) on survival and growth of larvae until total absorption of the yolk sac was demonstrated. Also, egg buoyancy at different salinities was established. Results indicate better hatching rates (70–78%) in temperatures ranging from 24 to 30 °C than at 20 °C. The notochord length of newly hatched larvae was inversely related to temperature and to the oil droplet diameter. Larvae that hatched at 20 °C were the longest and had the smallest oil droplet diameters and yolk sac volumes. The largest yolk sac volumes were observed at 26 and 28 °C. Times from fertilization to 50% hatch and hatching period duration decreased with increasing temperature. A higher larval growth rate to day 5 after fertilization was obtained at higher temperatures. Eggs maintained at 32‰ salinity showed the best hatching rates (80.7%). In the salinity growth trial, final notochord lengths of the larvae were inversely related to salinity. Eggs were buoyant at salinities above 34‰.

Sturgeon sperm was cryopreserved in liquid nitrogen or stored at 1–4 °C on a short-term basis. The objective was to establish reliable technologies for the conservation of certain endangered sturgeon species and for artificial reproduction in sturgeon farms. Cryopreseved sperm was diluted in buffered extenders (pH between 8 and 8.5) containing intracellular (dimethyl-sulfoxide (DMSO), methanol, ethylene glycol) or extracellular (saccharose, egg yolk) cryoprotectants. The concentration of intracellular cryoprotectants ranged from 5% to 20%. Tested freezing rates were in a range of 0.5–18.5 °C/min and the thawing rate between 15 and 40 °C/min for 5 to 6 s. Ten to eight percent of the sperm were motile after thawing. Fertilization rates up to 73–94% were reported for thawed sperm but in most cases, average values were 20%. The motility of non-frozen sperm was activated after few days of in vitro storage. Motility declined with time depending on oxygen availability in the milt, temperature (optimum near 0 °C) and pH; though some spermatozoa were still motile after several weeks of storage.

The present work optimizes procedures for artificial insemination of European catfish, Silurus glanis L., with goals to increase collection of sperm and hatching rate. We address technical aspects of immobilizing solution (IS), activation solution (AS), process of insemination, activation of gametes and elimination of eggs stickiness with a practical solution for artificial propagation in hatcheries. Spermiation was stimulated and could be sustained over a period of 1 month after weekly carp pituitary (CP) injection of 5 mg kg −1 b.w. Males produced significantly the largest quantity of sperm, 0.12–0.13·10 9 spermatozoa kg −1 b.w., after third and fourth injections. The best IS for sperm was determined as solution containing 170–200 mM NaCl, 30 mM Tris–HCl, pH 7 and activation solution for activation of gametes containing 17 mM NaCl, 5 mM Tris–HCl, pH 7. The ratio of 1:2 rather than ratio of 1:1 in the control batch (volume of activation solution in milliliters/grams of eggs) increased the hatchability up to 68%. The highest hatching success of 82–88% from four individual females were found for sperm number of 800–80,000 spermatozoa egg −1 , in comparison to 26% hatching at 80 spermatozoa egg −1 . The best time for application of alcalase enzyme for elimination of egg stickiness was 3 min after gamete activation at concentration of 20 ml enzyme l −1 with exposure of 2 min.

Cette etude resume l'optimisation des techniques pour la fecondation artificielle chez la carpe, y compris les ameliorations des solutions d'activation, du processus d'insemination, et d'elimination de la viscosite des oeufs. Pour obtenir de bons taux de fertilisation et d'eclosion, le rapport optimal se situe entre 8490 et 23 672 spermatozoides par oeuf, avec de l'eau du robinet dechloree. Le rapport optimal entre les oeufs (poids en grammes) et la solution d'activation (en millilitres) est definie comme etant de 1:1 a 1:2. Differentes concentrations de solutions d'activation, telles que NaCl de 0 a 34 mM (0 a 68 mOsmol kg -1 ) ne modifient pas les taux de fertilisation et d'eclosion. Une solution d'activation adoptee pour les spermatozoides de carpe (45 mM NaCl, 5 mM KCl, 30 mM Tris-HCl, pH 8) est comparee a d'autres solutions salines d'activation ; seule, la solution NaCl de 51 mM (102 mOsmol kg -1 ) diminue le taux de fertilisation et d'eclosion. La solution d'activation contenant 20 mM Tris-HCl a pH 9 augmente les taux de fertilisation et d'eclosion, comparee a l'eau du robinet dechloree de pH 7 ou a une solution d'activation de pH 6 et 7. La viscosite des oeufs est eliminee avec succes, par incubation dans l'alcalase DX (PLN 04715) avec deux etapes successives et differentes concentrations d'enzyme. La premiere etape, avec une concentration d'enzyme de 2 ml l -1 a ete appliquee de 8 a 20 min apres fertilisation. Dans une seconde etape, le meilleur temps d'application de l'alcalase, 20 ml l -1 , a ete de 45 et 60 s a 20 min apres fertilisation, conduisant a des taux de fertilisation et d'eclosion de 80-87 %. La α-chymotrypsine (EC 3.4.21.1. Merck) a ete aussi efficace pour l'elimination de la viscosite des oeufs mais avec des chances de succes beaucoup plus variables, dues aux differences environnementales, a la qualite de l'eau et aux differentes souches de carpe.

The European catfish Silurus glanis has been cultivated in extensive ponds in central and Eastern Europe in association with carps for more than 100 years. The total production of S. glanis in aquaculture from ten European countries (Austria, Bulgaria, Croatia, Germany, France, Hungary, Greece, Macedonia, Poland, Czech Republic and Romania) was 602 t in 1993 and presently is about 2 000 t. Production is increasingly from intensive pond culture or in heated/geothermal waters. The flesh is white, boneless, easy to prepare, and low in fat (6 to 8%). The taste is mild but is subject to off-flavour. The fish are filleted (yields about 40% without skin) and the flesh is cut into steaks or smoked). During the last 10 years, research in Czech Republic, France and other European countries have developed technologies for artificial reproduction, evaluated population genetics and addressed problems of conservation. Growth is higher in males and research is in progress to produce triploids fish. While propagation techniques have progressed, production remains low and the cost of production is still high. Thus availability is low and the product remains poorly known.

Although the common carp, Cyprinus carpio, has been cultivated for several thousand years and is produced in large quantities, research on reproduction has been very limited. Traditionally, spawning occurred naturally in situ in rearing ponds. In slightly improved methods large breeding ponds stocked with brood fish were devoted to reproduction with fry collection in autumn, or in small spawning ponds with collection of larvae a few days after hatching. Controlled reproduction in hatcheries started only in the 1950s. This paper reviews some of the basic work on carp sperm and describes the technologies of artificial reproduction. The sperm is of a primitive type with uncondensed chromatin and a small midpiece. As in most teleost fish, the sperm is immotile in the male genital tract and in the semen and is activated after release into fresh water. The initiation of motility is due to the decrease in osmotic pressure. The structure of the flagellum is rapidly disorganized in fresh water and sperm stop moving after 30 s. When dilution occurs in a 50 mM NaCl solution, the osmotic change is sufficient to initiate motility, but the flagellum is not disorganized and swimming lasts a few minutes. Motility depends mainly on endogenous ATP stores, about 12 nmol108 spermatozoa, and stops when 50–80% of the ATP is exhausted by hydrolysis. The procedure of artificial insemination includes collection of gametes (from “hypophysised” males and females), mixing in an extender (45 mM NaCl, 5 mM KCl, Tris 2.5 mM, glycine 19 mM, pH 8) in a ratio 1 litre of eggs to 1 litre of diluent and 1 ml of semen. Sperm motility can be triggered during collection by contamination of the semen with urine; this could interfere with fertilization and, if present, urine should be discarded by pouring out the top part of the tube. The sticky layer of the egg is removed by adding a “dissolving solution” (20 g urea/1 + 4g NaCl/1 of water) or milk (diluted 15 in water) to the fertilized egg and stirring. One hour later the swollen eggs are transferred to incubators (usually Zug bottles, sometimes 200 litre circular jars).

Increased interest in the domestic cat, both as a companion animal and as a model for rare felid species, has led to augmented research focusing on reproductive processes in this species. In the male, primary attention has been aligned to investigations of gamete retrieval and cryopreservation. Epididymal sperm cells obtained from reproductive tracts after neutering (simulating posthumous collection) do capacitate, as evidenced by attachment to homologous zonae with 30 min and maximum attachement after 4 h. Following cryopreservation, motility is observed, but is attenuated compared with pre-freeze values by 30–40%. However, this information does not correlate with fertilizing ability, as an average of less than 20% of the acrosomes remain intact post-thaw and fertilization rates are less than 2%. Investigations of non-invasive endocrine techniques have centered on the female and new methods for monitoring fecal steroids have been described and adapted to species such as the cheetah, lion, tiger, bobcat and serval. The most intense area of research has been development of artificial breeding techniques. Recently, a 50% success rate with laparoscopic artificial insemination has been demonstrated if semen deposition occurs after ovulation, whereas pregnancy rates are severely attenuated when insemination is performed before the ovulatory process. This technique now has been adapted to the leopard cat, cheetah, tiger and puma, with the birth of one offspring from each species. In vitro fertilization also has received much attention and results in 84–85% fertilization rates using Ham's F-10 medium with PMSG/hCG-stimulated cocytes (84 h between injections). Adaptation to non-domestic felids has resulted in the birth of one litter of tiger cubs and Indian desert cat. Salvage of female gametes by in vitro oocyte maturation and fertilization is a recently exploited area of research for the domestic cat. Approximately 50% of all oocytes placed in culture develop to the metaphase II stage, with gonadotropin-supplemented medium providing superior conditions for maturation. These oocytes are capable of undergoing fertilization (range, 11–36%), and embryos derived completely from male and female rescued gametes have been produced (fertilization rate, approximately 20%). Because of the enormous progress made with domestic cat artificial reproduction, the developed methods (e.g. semen cryopreservation, artificial insemination, in vitro fertilization, gamete salvage) will undoubtedly serve as important tools for strategic management of endangered species populations.

Global population pressure has resulted in the depletion of many natural resources with the subsequent compromise of the natural habitat which supports much of the world's wildlife. Zoos have become "the custodians of the irreplaceable products of nature's evolution", and zoo management philosophy has changed in focus from one of exhibition and entertainment to one which embraces education, research and conservation. More intensive management practices at the Houston Zoological Gardens have made possible the adaptation of artificial reproduction techniques to a pair of southern white rhinoceroses ( Ceratotherium simum simum). Both animals have been trained to enter a chute for blood collection, rectal palpation, external massage and olfactory stimulation. Regular collection of urine and nasal mucus samples for steroid evaluation and vaginal smears for cytological examination provide a further basis for monitoring ovarian activity in the female. Daily behavioral observations are correlated with other parameters in order to establish the normal reproductive cycle. The ultimate goal is successful artificial insemination of the white rhinoceros.

A comparison between gilthead seabream ( Sparus aurata ) obtained by artificial reproduction and wild ones is reported for extensive, semi-intensive and intensive rearing conditions. Growth, survival, food conversion factor and production were similar for both populations. A considerable percentage (19–48%) of deformed individuals was found in the artificial fish population. The condition factor of these fish was not suitable for investigating their “well-being”. If population type is discarded, significant growth differences are related more to the three culture systems than to artificial or wild populations.

Two experiments were carried out to study the effect of different feeding levels and feeding frequencies in the culture of Clarias lazera (C. & V.) fingerlings from 0.5 to 10 g. It was found that 0.5 g fingerlings could reach an average weight of about 10 g in 3–4 weeks when they were fed continuously for 24 h per day or 12 h at night at a daily rate of 8% of the biomass or more. A feeding level of 10% of the biomass appeared to be optimal, from the point of hygiene, growth rate and feed utilization parameters. Continuous feeding during part or all of the day clearly gave better growth than feeding two or four defined meals per day. When these preferred feeding regimes were practised, the feed conversion (wet weight) ranged from 0.65 to 0.97, the protein efficiency ratio from 2.14 to 3.04 and the productive protein value from 0.34 to 0.47. These values compare favourable with values reported for other cultured species, indicating that C. lazera fingerlings are efficient feed convertors. It could be calculated that, when the rations are readjusted only once a week, the high growth rates occurring in these experiments result in a considerable difference between the designated feeding rates and the actual feeding rates.

Artificial reproduction techniques were applied to individual animals not capable of natural reproduction. All individuals represented endangered species. An aged and debilitated Arabian oryx was treated with prostaglandin F2 α (PG) and pregnant mare serum gonadotropin (PMSG) prior to euthanasia. She responded to the treatment with mild superovulation. Four follicular oocytes were recovered, all of which subsequently matured in vitro and were frozen. A group of three Soemmering's gazelles, including a crippled male and a three-legged female, was evaluated for reproductive potential. The male was electro-ejaculated and determined to be producing normal sperm; semen was frozen. Estrus was induced and synchronized in the two females with PMSG and PG prior to the introduction of the male. Copulation attempts were unsuccessful. A second modified estrus induction attempt did not result in behavioral estrus in either female. The handicapped female was subsequently superovulated with PMSG before euthanasia. Four oocytes were recovered but failed to mature in vitro. Two Przewalski's horse mares are not able to mate naturally due to chronic laminitis. Preliminary studies have been undertaken to determine, through ultrasound examination, the estrous cycles and ovarian dynamics of the two horses, in preparation for artificial insemination and embryo transfer. Cycle length and variability, ovarian response to PG and rate of follicle growth have been determined to be similar to the domestic horse.