The challenge of assisted reproduction for conservation of wild felids - A reality check.

Threats to the Earth's biodiversity are increasing exponentially, driven by human population growth and resource consumption. As many as one million wildlife species may disappear within the next few decades due to this human-induced extinction event. This represents our current reality and has profound implications for wildlife conservation. Within this context, application of assisted reproductive technology (ART) to conservation management is unlikely to mitigate broad-scale species loss, but for select species, such as wild cats, ART may determine if populations survive or disappear. In North American and European zoos, 20 of the world's 38 wild felid species are managed within structured breeding programs, but most are not sustainable with natural breeding alone. Zoo-based breeding programs are facing tenuous futures due to triage-based responses to this growing sustainability crisis. Theoretically, ART could benefit conservation management, but only by recognizing and addressing its present challenges. The application of ART to wildlife has been rarely successful, with only 62 mammal species (including 15 cat species) ever propagated by AI, and just 35 of these species (6 cats) reproduced following frozen semen AI. Even this most basic form of ART has a minimal impact on wildlife sustainability. The drivers of this deficit include lack of species-specific reproductive knowledge and limited access to animals for study, but also is exacerbated by a science-conservation disconnect that attempts to apply advanced reproductive technologies to species in which basic ART remains unproven. For a few felid species, these scientific challenges have been overcome and AI with frozen semen is becoming feasible as a practical management tool; for other felids, further research is needed. Non-scientific issues also impair our ability to use ART to implement global management plans. Political dysfunction, regulatory barriers and societal indifference create inertia that interferes with achieving meaningful progress in applying ART to wildlife. Collectively, these challenges may seem insurmountable but human resiliency is essential if we are to resolve these issues in a systematic manner. It will require expanding collaborative efforts substantially and intensifying efforts to conserve wildlife species that are literally running out of time. Our goal is to create a new reality that includes a sustainable future for wild felids and other imperiled wildlife species.

Recovering an endangered frog species through integrative reproductive technologies.

The establishment and management of ex situ breeding and assurance populations around the globe are meant to provide short-term solutions to the formidable loss of amphibian diversity presently occurring. Large multi-scaled facilities, such as zoos and aquariums, can provide the infrastructure to safeguard species and populations. However, often even large, economically viable facilities lack the knowledge to efficiently cater to the plethora of environmentally controlled physiological strategies that amphibians possess. Anurans present a class of amphibians that have often been viewed as easy to maintain ex situ. However, while adult survival may be relatively successful it is rarely accompanied by good reproductive output, health, and fitness. Even more conspicuous is the low survivorship of offspring produced ex situ once they are translocated back into the wild. The mountain yellow-legged frog (R. muscosa) ex situ breeding program EBP is a prime example of the challenges that amphibians EBPs face. Although more research is needed, the R. muscosa program has increased reproductive output and health of its colony by incorporating reproductive technologies and strategic genetic management in conjunction with a greater understanding of the species' natural history, to produce and translocate viable animals each year. This paper highlights the EBPs past decade of research featuring the program's contribution to building empirical, multidisciplinary approaches that boost the robustness of an endangered species, by safeguarding existing genetic diversity and maximizing fitness and survival outcomes.

Progress and challenges in developing organoids in farm animal species for the study of reproduction and their applications to reproductive biotechnologies.

Within the past decades, major progress has been accomplished in isolating germ/stem/pluripotent cells, in refining culture medium and conditions and in establishing 3-dimensional culture systems, towards developing organoids for organs involved in reproduction in mice and to some extent in humans. Haploid male germ cells were generated in vitro from primordial germ cells. So were oocytes, with additional support from ovarian cells and subsequent follicle culture. Going on with the female reproductive tract, spherical oviduct organoids were obtained from adult stem/progenitor cells. Multicellular endometrial structures mimicking functional uterine glands were derived from endometrial cells. Trophoblastic stem cells were induced to form 3-dimensional syncytial-like structures and exhibited invasive properties, a crucial point for placentation. Finally, considering the embryo itself, pluripotent embryonic cells together with additional extra-embryonic cells, could self-organize into a blastoid, and eventually into a post-implantation-like embryo. Most of these accomplishments have yet to be reached in farm animals, but much effort is devoted towards this goal. Here, we review the progress and discuss the specific challenges of developing organoids for the study of reproductive biology in these species. We consider the use of such organoids in basic research to delineate the physiological mechanisms involved at each step of the reproductive process, or to understand how they are altered by environmental factors relevant to animal breeding. We evaluate their potential in reproduction of animals with a high genetic value, from a breeding point of view or in the context of preserving local breeds with limited headcounts.

Ethics and animal reproductive technologies.

This paper offers a framework to help animal scientists engage in critical thinking about their own practices. Its objective is to reinforce their ability to participate in debates and discussions about the ethics surrounding the use of modern animal reproductive technologies (ART). This will be achieved first by exploring some of the most important philosophical conceptualizations of animals in Western philosophy, which are shaping the way humans interact with them. Then, we will analyse whether modern ART constitute ethically significant innovations in comparison with more traditional animal breeding practices, or whether they stand in continuity with the latter. This will be followed by a review some of the most important ethical issues with modern ART, where human, animal welfare, environmental and socio-economic issues will be discussed.

A cascade of dams affects fish spatial distributions and functional groups of local assemblages in a subtropical river

Dams reduce the longitudinal connectivity of rivers and thereby disrupt fish migration and the spatial distribution of species, impacts that remain poorly studied for some Neotropical rivers from mega-diverse basins. We investigated the spatial distribution of fish species with different trophic and movement/reproductive/size characteristics to assess how functional groups have responded to a cascade of dams on the Uruguai River in southern Brazil. Fish abundance, biomass, and species composition were evaluated at eight locations along the longitudinal gradient. The fish assemblage in the upper stretch was mainly characterized by small and medium-sized species at higher trophic levels, whereas the sites located furthest downstream displayed more medium and large-sized species, including many carnivorous species. Species with high fecundity, seasonal migrants, and catfishes with internal fertilization were common in the river´s middle and lower reaches. Detritivorous species dominated areas distant from the dams. Overall, functional diversity of local fish assemblages was greater in lower reaches. The cascade of dams has impacted the distribution of functional groups of local fish assemblages of Uruguai River. The alteration of functional groups in upper reaches of the river has potential consequences for ecosystem processes and services, such as nutrient cycling and fisheries.(AU)

As barragens reduzem a conectividade longitudinal dos rios e interrompem a migração e a distribuição espacial das espécies, configurando impactos pouco estudados para alguns rios Neotropicais megadiversos. Investigamos a distribuição espacial de espécies de peixes com diferentes características tróficas e reprodutivas para avaliar como grupos funcionais responderam a uma cascata de reservatórios no rio Uruguai, sul do Brasil. Abundância de peixes, biomassa e composição de espécies foram avaliadas em oito locais ao longo do gradiente longitudinal. A assembleia de peixes no trecho superior foi caracterizada principalmente por espécies de pequeno e médio porte em níveis tróficos mais elevados, enquanto que os ambientes localizados mais a jusante apresentaram mais espécies de médio e grande porte, incluindo muitas espécies carnívoras. Espécies com alta fecundidade, migradores sazonais e bagres com fertilização interna foram comuns no curso médio e inferior. Espécies detritívoras dominaram áreas distantes das barragens. A diversidade funcional das assembleias de peixes locais foi maior nas partes inferiores. A cascata de barragens impactou a distribuição dos grupos funcionais das assembleias de peixes locais no rio Uruguai. A alteração da diversidade funcional no curso superior do rio tem consequências potenciais para a dinâmica e serviços do ecossistema, como ciclagem de nutrientes e pesca.(AU)

Current Reproductive Technologies Impacting Equine Embryo Production.

Numerous reproductive technologies have been developed in the past several decades, which have dramatically changed the way mares are bred. This review will focus on embryo recovery and transfer, cooled-shipped embryos, embryo freezing, oocyte freezing, oocyte collection and transfer, intracytoplasmic sperm injection (ICSI), and sexed semen. Embryo transfer procedures have been constant for many years and the costs have not changed. The major change has been the ability to store embryos at 5 C for 12-24 hours and transport them to recipient stations. Embryo freezing has become more common using the technique of vitrification of embryos >300 µm or deflating embryos >300 µm before freezing. Oocyte vitrification has resulted in poor pregnancy rates although the technique works well in women. The ability to collect oocytes from mares and fertilize them by sperm injection has revolutionized the veterinarian's approach to infertility in the mare and/or stallion. A transvaginal approach can be used to collect oocytes from preovulatory follicles and unstimulated follicles 5-25 mm in size. Although traditional in vitro fertilization does not work well in the horse, ICSI can be used to produce blastocysts which, upon nonsurgical transfer into recipients, provide a pregnancy rate similar to fresh embryos collected from donor mares. Sorting sperm by flow cytometry into X- and Y-bearing spermatozoa has been shown to provide about a 50% pregnancy rate with freshly sorted sperm but only 12% with sorted, frozen/thawed stallion sperm. It is likely that more advanced reproductive techniques will be developed in the future. Their acceptance will depend on how well they work, perceived need, cost, and, to some extent, the breed associations.

Using Vaginal Impedance Measurement to Identify Proestrus in Rats Given Luteinizing Hormone Releasing Hormone (LHRH) Agonist.

Vaginal cytology is the most common method of monitoring the estrous cycle in rats; however, this test requires specific technical training and can be subject to interpretation. Vaginal impedance offers a quicker and less technically challenging alternative and has been used successfully to identify estrus in normally cycling breeder rats. We hypothesize that vaginal impedance can also be used to stage the estrous cycle in rats that have been given luteinizing hormone releasing hormone (LHRH) for timed mating. Vaginal impedance measurements and vaginal cytology were performed in LHRH-primed female rats (n = 36) at the expected peak of proestrus and paired with proven stud males. Breeding success was determined by gross necropsy to detect embryo implantation sites in the female rats. We found that the predictive rates of vaginal cytology and impedance measurement for proestrus were similar; however, both methods resulted in high proportions of false positive and false negative determinations (28% and 31%, respectively). We further hypothesized that females respond to LHRH at variable rates, resulting in variable times of peak proestrus. To test this, vaginal impedance measurements were performed multiple times throughout the expected day of proestrus in LHRH-primed female rats (n = 36). Females were either paired with a male 24 h after reaching the proestrus threshold (n = 18) or paired according to our standard protocol at 1300 h on the day after the expected proestrus (n = 18). Sequential measurements reduced false positive and negative rates (14% and 8%, respectively). Pregnancy rates did not differ based on the time of pairing during expected estrus. Overall, we determined vaginal impedance can be more successful than vaginal cytology at identifying proestrus in the rat, but only if multiple measurements are taken.

Evaluating amphibian biobanking and reproduction for captive breeding programs according to the Amphibian Conservation Action Plan objectives.

The Amphibian Conservation Action Plan (ACAP), published in 2007, is a formal document of international significance that proposed eleven relevant actions for global amphibian conservation. Action seven of the ACAP document addresses the use of amphibian captive programs as a conservation tool. Appendix material under this action explores the potential use of Genome Resource Banking (biobanking) as an urgently needed tool for these captive programs. ACAP proposed twelve objectives for Genome Resource Banking which exhibit little emphasis on reproduction as a vital underlying science for amphibian Captive Breeding Programs (CBP's). Here we have reassessed the original twelve ACAP objectives for amphibian reproduction and biobanking for CBP's as a contribution to future ACAP review processes. We have reviewed recent advances since the original objectives, as well as highlighted weaknesses and strengths for each of these objectives. We make various scientific, policy and economic recommendations based on the current reality and recent advances in relevant science in order to inform future ACAP towards new global objectives. The number of amphibian CBP'S has escalated in recent years and reproductive success is not always easily accomplished. Increases in applied and fundamental research on the natural history and reproductive biology of these species, followed by the appropriate development and application of artificial reproductive technologies (ART's) and the incorporation of genome resource banks (GRB's), may turn CBP's into a more powerful tool for amphibian conservation.

Genetic evaluation of milking buffaloes (Bubalus bubalis): bull ranking / Avaliação genética de búfalas leiteiras (Bubalus bubalis): classificação de touros

The objective of this study was to evaluate genetic aspects related to production and reproductive efficiency of Murrah and Mediterranean buffaloes and their crosses. A ranking of bulls from Embrapa Eastern Amazonia was also composed to guide assisted mating. Birth records of 2,322 Murrah, Mediterranean, and crossbred buffaloes from the Embrapa Eastern Amazon herd, from 1953 to 2013, as well as information on production and reproductive traits were used. Genetic analyzes were performed in the WOMBAT software using the animal model with two-trait analysis. While heritability (h2) for total milk production (TMP) and fat milk percentage (F) were generally high, for reproductive traits h2 tended to be low. Genetic correlations for TMP with the other traits were low and negative, except for TMP with calving interval (CI) and service period (SP) in the Mediterranean breed and with age at first calving (AFC) and SP in crossbred, which were positive and high. Bull 1001 had high predicted transmitting ability (PTA) for TMP, so it should transmit a greater volume of milk to his offspring, although it had a lower PTA for F. There was sufficient variability within the herd to work with genetic management for both production and reproductive efficiency.(AU)

O objetivo deste estudo foi avaliar os aspectos genéticos relacionados à produção e à eficiência reprodutiva de búfalas das raças Murrah, Mediterrâneo e suas cruzas. Uma classificação de touros da Embrapa Amazônia Oriental também foi composta para orientar os cruzamentos assistidos. Foram utilizados 2.322 registros de nascimento de búfalas das raças Murrah, Mediterrâneo e cruzadas do rebanho da Embrapa Amazônia Oriental, de 1953 a 2013, bem como características produtivas e reprodutivas. As análises genéticas foram realizadas pelo software Wombat, utilizando-se o modelo animal com análise de duas características. Enquanto a herdabilidade (h 2 ) para a produção total de leite (PTL) e para a porcentagem de gordura (G) foi alta, para as características reprodutivas a h 2 tendeu a ser baixa. As correlações genéticas da PTL com as demais características foram baixas e negativas, exceto para a PTL com intervalo entre partos (IP) e período de serviço (PS) na raça Mediterrâneo e com idade ao primeiro parto (IPP) e PS nas cruzadas, que foram positivas e altas. O touro 1001 apresentou alta capacidade de transmissão predita (CTP) para a PTL, então deve transmitir um maior volume de leite para seus descendentes, embora com um menor conteúdo transmissível de CTP para G. Portanto, existe variabilidade suficiente dentro do rebanho para trabalhar com o manejo genético para a produção e a eficiência reprodutiva.(AU)

Efeito da técnica de coleta e do estágio do ciclo estral na recuperação de oócitos de boa qualidade em felinos e caninos domésticos / The effect of recovery technique and stage of estrous cycle on the recovery of good quality oocytes in domestic felines and canines

A obtenção de oócitos de boa qualidade é essencial para o sucesso de diversas biotécnicas reprodutivas. Objetivou-se determinar o efeito de duas técnicas na recuperação de oócitos de boa qualidade em gatas e cadelas em diferentes estágios reprodutivos. Foram utilizados 43 pares de ovários de gata e 35 de cadela após realização da ovariosalpingohisterectomia eletiva. A fase do ciclo estral foi classificada em inativa, folicular ou luteal. Os ovários da fase folicular foram divididos em três grupos: PUN) punção dos folículos com agulha; PUN+FAT) fatiamento do mesmo ovário já puncionado; e FAT) fatiamento do segundo ovário. Os ovários das fêmeas em fase luteal e inativa foram submetidos ao FAT. Foram obtidos no total 974 oócitos (~23/animal) nas fêmeas felinas e 940 (~27/animal) nas caninas. O fatiamento recuperou número superior (P<0,05) de oócitos. Não houve diferença (P>0,05) entre as técnicas de coleta na qualidade de estruturas recuperadas. A quantidade de oócitos recuperados em cada fase foi similar (P>0,05). Contudo, a fase inativa foi superior à luteal (P<0,05) e semelhante à folicular na quantidade de oócitos de boa qualidade em gatas e não houve diferença em cadelas. Conclui-se que o fatiamento recupera maior quantidade de oócitos, não influenciando em sua qualidade. As fases inativa e folicular recuperam maior quantidade de oócitos de boa qualidade em gatas e não afetam a recuperação em cadelas. Portanto, para otimizar o uso das biotecnologias, deve-se levar em consideração o estágio do ciclo estral em fêmeas felinas e a técnica de coleta utilizada na recuperação de oócitos.

The recovery of good quality oocytes is essential for the success of various reproductive biotechniques. The aim of this study was to determine the effect of two techniques on the recovery of good quality oocytes in queens and bitches at different reproductive stages. A total of 43 pairs of ovaries of queens and 35 of bitches after elective ovariosalpingohisterectomy were performed. The estrous cycle phase was classified as inactive, follicular or luteal. The ovaries of the follicular phase were allocated into three groups: PUN) puncture of the follicles with a needle; PUN + SLI) slicing of the same ovary already punctured; and SLI) slicing of the second ovary. The ovaries of luteal and inactive females were submitted to SLI. A total of 974 oocytes (~23/animal) were obtained in feline females and 940 (~27/animal) in canines females. The SLI technique recovered superior number (P<0.05) of oocytes. There was no difference (P>0.05) between the collection techniques in the quality of recovered structures. The number of oocytes recovered in each phase was similar (P>0.05). However, the inactive phase was higher than luteal (P<0.05) and similar to the follicular phase in the quantity of good-quality oocytes in queens and there was no difference in bitches. In conclusion, it is preferable to perform the slicing technique to recover more oocytes in both species. Moreover, in queens it is possible to obtain good quality oocytes in the inactive phase and in bitches the estrous cycle phase does not influence the quality.

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.

Effect of Spirulina and Vitamin E on Reproduction and in vitro Embryo Production in Heat-stressed Rabbits.

BACKGROUND AND OBJECTIVE: High ambient temperature can cause heat stress and evokes a combination of change in blood biochemicals and reproduction of rabbit. This study targeted to investigate the effect of Spirulina platensis, vitamin E and their combination on in vivo and in vitro reproductive performance and some physiological and health indicators of heat stressed rabbit does. MATERIALS AND METHODS: Nili-parous rabbit does (n = 80) were allocated to 4 groups. Does in the 1st group were fed commercial complete feed diet, while those in the 2nd, 3rd and 4th were fed complete feed diet with Spirulina platensis (300 mg kg-1), vitamin E (100 mg kg-1 diet) and Spirulina platensis+vitamin E kg-1 diet, respectively. All does were naturally mated with fertile bucks (5 bucks/group). RESULTS: The does in the 2nd group showed significantly (p<0.05) better reproductive performance (conception rate, kindling rate and litter size), lipid profile (total lipids, cholesterol, triglycerides, high and low density lipoproteins, antioxidant capacity (total antioxidant capacity, glutathione, malondialdehyde, glutathione peroxidase, glutathione S-transferase, superoxide dismutase and catalase), immunity (lysozyme, IgG and IgM), ovulatory response (corpora lutea number and ovulation rate), embryo quality and hatched blastocysts production with higher cell number and inner cell mass as compared to other groups. CONCLUSION: Dietary supplementation with Spirulina platensis (300 mg kg-1 diet), in comparing with vitamin E (100 mg kg-1 diet) or their combination at the same levels, had positive impact on reproductive performance of rabbit does used in breeding program under heat stress condition in Egypt.

The Role of Reproductive Technologies in Amphibian Conservation Breeding Programs.

Anthropogenic environmental change has led to unprecedented rates of species extinction, presenting a major threat to global biodiversity. Among vertebrates, amphibians have been most severely impacted, with an estimated 41% of species now threatened with extinction. In response to this biodiversity crisis, a moral and ethical obligation exists to implement proactive interventionist conservation actions to assist species recovery and decelerate declines. Conservation breeding programs have been successfully established for several threatened amphibian species globally, aiming to prevent species' extinction by maintaining genetically representative assurance colonies ex situ while providing individuals for population augmentation, translocation, and reestablishment in situ. Reproductive technologies have enormous potential to enhance the propagation and genetic management of threatened species. In this review, we discuss the role of reproductive technologies in amphibian conservation breeding programs and summarize technological advancements in amphibian hormone therapies, gamete storage, and artificial fertilization.

Been There, Done That: A Practical Primer for Veterinarians Considering Inclusion of Small Animal Theriogenology Services in Their Practice.

Broadening your scope of practice to include theriogenology services offers a myriad of advantages. Theriogenology services are profitable, offer new revenue streams, and optimize the use of support staff and hospital. Offering reproductive services sets your practice apart from competitor practices. Breeder clients are demanding but loyal and return for repeat services; they also request and follow recommendations for "high-end" services. Your theriogenology clients often refer locally placed puppies and kittens to you for primary care and you gain new general practice clients. And it is fun!

A 100-Year Review: Reproductive technologies in dairy science.

Reproductive technology revolutionized dairy production during the past century. Artificial insemination was first successfully applied to cattle in the early 1900s. The next major developments involved semen extenders, invention of the electroejaculator, progeny testing, addition of antibiotics to semen during the 1930s and 1940s, and the major discovery of sperm cryopreservation with glycerol in 1949. The 1950s and 1960s were particularly productive with the development of protocols for the superovulation of cattle with both pregnant mare serum gonadotrophin/equine chorionic gonadotrophin and FSH, the first successful bovine embryo transfer, the discovery of sperm capacitation, the birth of rabbits after in vitro fertilization, and the development of insulated liquid nitrogen tanks. Improved semen extenders and the replacement of glass ampules with plastic semen straws followed. Some of the most noteworthy developments in the 1970s included the initial successes with in vitro culture of embryos, calves born after chromosomal sexing as embryos, embryo splitting resulting in the birth of twins, and development of computer-assisted semen analysis. The 1980s brought flow cytometric separation of X- and Y-bearing sperm, in vitro fertilization leading to the birth of live calves, clones produced by nuclear transfer from embryonic cells, and ovum pick-up via ultrasound-guided follicular aspiration. The 20th century ended with the birth of calves produced from AI with sexed semen, sheep and cattle clones produced by nuclear transfer from adult somatic cell nuclei, and the birth of transgenic cloned calves. The 21st century has seen the introduction of perhaps the most powerful biotechnology since the development of artificial insemination and cryopreservation. Quick, inexpensive genomic analysis via the use of single nucleotide polymorphism genotyping chips is revolutionizing the cattle breeding industry. Now, with the introduction of genome editing technology, the changes are becoming almost too rapid to fully digest.

Performance reprodutiva de leitoas submetidas à inseminação artificial pós-cervical / Reproductive performance of gilts submitted to post-cervical artificial insemination

O objetivo deste trabalho foi avaliar o desempenho reprodutivo de nulíparas submetidas à inseminação artificial pós-cervical (IAPC) comparada à inseminação artificial tradicional (IAT). Foram avaliados ocorrência de sangramento, ocorrência de refluxo, dificuldade no transpasse da cérvix e total de células refluídas até 30 minutos após inseminação. Fêmeas submetidas à IAPC (n=279) foram inseminadas com doses de 1,5 x 109 diluídas em 45mL, e fêmeas submetidas à IAT (n=273) inseminadas com doses de 2,5 x 109 em 80mL. O transpasse da cérvix foi possível em 91,04% (254/279) das leitoas. A dificuldade no transpasse foi de 41,58% (116/279), não comprometendo o desempenho reprodutivo (P>0,05). Presença de sangramento não afetou a taxa de parto nem o número de leitões nascidos para ambos os tratamentos (P>0,05). O percentual de espermatozoides presentes no refluxo foi maior na IAT, não sendo observada diferença no tamanho de leitegada de acordo com o percentual de espermatozoides no refluxo (P>0,05) e no número de leitões nascidos totais (11,63 e 11,81) entre os tratamentos IAT e IAPC, respectivamente. Pode-se realizar IAPC em leitoas sem causar redução no desempenho, utilizando-se doses com 1,5 x 109 células espermáticas.(AU)

The objective of this study was to evaluate the reproductive performance of gilts subjected to post-cervical artificial insemination (PCAI) compared to traditional artificial insemination (TAI). We also evaluated the degree of difficulty in bypassing the cervix, time required to perform the insemination, presence of bleeding after insemination, semen backflow, as well as the volume and the total reflow cells 30 minutes after insemination. Gilts submitted to PCAI (n = 279) were inseminated with 45 mL doses of 1.5 x 109 sperm cells and the ones submitted to TAI (n = 273) were inseminated with 80 mL doses with 2.5 x 109 cells. The bypassing of the cervix was possible in 91.04% (254/279) of gilts. The difficulty bypassing the cervix in at least one of the gilt's PCAI procedures happened with 41.58% (116/279) of the females, but it did not affect reproductive performance (P>0.05). The presence of bleeding after insemination did not affect the farrowing rate and total number of piglets born for both treatments (P>0.05). The average time needed to carry out the PCAI was 1.47 minutes and the TAI was 4.04 minutes. The percentage of sperm present in the reflux was higher in TAI than the PCAI, but no correlation was found between litter size and the percentage of sperm in reflux (P>0.05) and the total number of piglets born (11.63 and 11.81) between TAI and PCAI treatments, respectively. Thus, it is possible to perform the post-cervical artificial insemination in gilts without causing a reduction in reproductive performance, using doses with a concentration of 1.5 x 109 sperm cells.(AU)

Effect of dilution in sperm maturation media and time of storage on sperm motility and fertilizing capacity of cryopreserved semen of sex-reversed female rainbow trout.

Masculinized females, also called neomales or sex-reversed females have a male phenotype but retain the female genotype (XX). Therefore, all spermatozoa produced in their functional testes carry an X chromosome, which is desired for the production of all-female rainbow trout populations. Semen of sex-reversed female rainbow trout is of low quality and in vitro maturation is required, which includes dilution of sperm suspensions with specially formulated maturation solutions. The aim of this study was to determine the effect of dilution in different maturation media on sperm quality (sperm motility characteristics and fertilizing capacity) of frozen/thawed sperm of sex-reversed female rainbow trout. The effect of time of post-thaw storage (0, 15, 60 and 120min) on semen quality was also tested. Sperm motility parameters and fertilization rate at the eyed and hatching stages were assessed for post-thaw semen diluted in different media. The cryopreservation procedure resulted in high post-thaw sperm motility of about 57% and did not differ from fresh semen. Unexpectedly, maturation media decreased sperm activation capacity immediately after dilution; however, sperm motility increased over time. Fertilization rates of frozen/thawed semen were high (71-87%) and did not differ significantly between experimental variants at any of tested periods of storage. Our results demonstrated that the effect of the maturation media on frozen/thawed sperm is different from that of fresh sperm. The progressive increase in post-thaw sperm motility in maturation media can potentially be applied to routine hatchery practice.

Reproduction, feeding and migration patterns of Prochilodus nigricans (Characiformes: Prochilodontidae) in northeastern Ecuador

The black prochilodus, Prochilodus nigricans, is an important food fish distributed throughout aquatic habitats of the Ecuadorian Amazon. We sampled gonad weight, body condition, and feeding of this species to address the question of whether changes in these biological parameters are related to hydrological variation and migration patterns. High frequency of empty stomachs and poor body condition corresponded to migration periods. Gonad condition analysis revealed a single, discrete spawning period in April (end of rising water season). We synthesized our own and published observations on migration patterns of black prochilodus and presumptive factors that influence those movements. Mature individuals make lateral migrations from peripheral flooded habitats to large white water rivers to spawn. Eggs and larvae drift downstream to nurseries at least into Peru and perhaps into Brazil; there do not appear to be nursery habitats in Ecuador. After 6-18 months, they migrate upstream, recruiting to parental stocks. Long-distance migrations of black prochilodus and similar fishes in Neotropical rivers disobey political boundaries. Conservation and management of such migratory fishes, thus, requires international co-operation and integrated conservation efforts.(AU)

El bocachico, Prochilodus nigricans, es una fuente importante de proteínas, distribuido en hábitats acuáticos de las tierras bajas de la Amazonía ecuatoriana. Se muestreo el peso de las gónadas, la condición corporal, y alimentación de esta especie para determinar la relación de estos parámetros biológicos con la variación hidrológica y patrones migratorios. Una frecuencia alta de estómagos vacíos y la pobre condición corporal corresponde a períodos de migración. El análisis del estado de las gónadas reveló un corto periodo de desove en abril (fin de la temporada de aumento del río). Hemos sintetizado nuestras observaciones propias y publicadas sobre migraciones del bocachico, así como los factores que presumiblemente influyen en estos movimientos. Los peces adultos migran lateralmente desde hábitats periféricos inundados hasta ríos de aguas blancas para desovar. Los huevos y larvas flotan aguas abajo hasta llegar a sitios de crianza en Perú y posiblemente en Brasil; no parece haber hábitats de crianza en Ecuador. Después de 6-18 meses, los bocachicos migran aguas arriba, reclutando a las poblaciones parentales. Las migraciones a larga distancia del bocachico y peces similares en ríos Neotropicales desobedecen las fronteras políticas. Una gestión apropiada de este recurso requiere de cooperación internacional y esfuerzos integrados de conservación.(AU)

Efeito de diferentes concentrações de melatonina em espermatozoides de carneiros sobre estresse oxidativo após criopreservação / Effect of different concentrations of melatonin to ram spermatozoa on oxidative stress after cryopreservation

Objetivou-se avaliar o efeito da adição de diferentes concentrações de melatonina no sêmen diluído de carneiros após criopreservação. Foram coletados 10 ejaculados de três carneiros adultos (n=30), por meio de vagina artificial para ovinos. Os ejaculados coletados foram diluídos em Tris-Gema de ovo, para a concentração final de 200x106 sptz/mL, mantidos em banho maria a 32°C, e a melatonina adicionada conforme os tratamentos: Controle; 100pM; 100nM; 100µM e 1mM de melatonina. Então, as amostras foram resfriadas em câmara fria a 5°C por duas horas, envasadas em palhetas de 0,5 mL e lacradas. Logo após, foram acondicionadas sob vapores do nitrogênio liquido, por 15 minutos, a 8cm da lâmina líquida e congeladas com nitrogênio líquido. As amostras foram analisadas quanto à motilidade espermática, integridade da membrana plasmática, membrana acrossomal, atividade mitocondrial, quantificação do estresse oxidativo e a capacidade de ligação. As variáveis foram submetidas à análise de variância e as médias foram comparadas pelo teste de Tukey a 5% de probabilidade. A motilidade total e progressiva dos espermatozoides descongelados foi maior nas amostras tratadas com 100pM de melatonina (62,99 e 45,07% respectivamente; P<0,05) quando comparado aos demais tratamentos. A adição das diferentes concentrações de melatonina no sêmen diluído, com exceção da concentração de 1 mM, apresentou maior percentual de células com membrana plasmática íntegra, quando comparadas com o controle (P<0,05). O percentual de espermatozoides com integridade da membrana do acrossoma foi maior no sêmen tratado com 100 pM de melatonina (P<0,05) do que nos demais tratamentos. A alta atividade mitocondrial foi maior nos espermatozoides tratados com 100 pM de melatonina (69,30%; P<0,05). A adição de 100 nM de melatonina reduziu a quantidade de TBARS após a criopreservação (2,84; P<0,05) quando comparado aos demais tratamentos. Após o descongelamento, o número de espermatozoides que se ligaram à membrana perivitelina foi maior nos tratados com 100 pM de melatonina (155,73; P<0,05). Portanto, a adição de melatonina no sêmen diluído pode ser útil para aperfeiçoar a criopreservação do sêmen de ovinos, melhorando as taxas de fertilização por meio da inseminação artificial.(AU)

The aim was to evaluate the effect of adding different concentrations of melatonin in ram semen diluted after cryopreservation. Ten ejaculates were collected0 from three adult ram (n=30) by means of artificial vagina for sheep. The collected samples were diluted in Tris-egg yolk, to a final concentration 200x106 sptz/mL kept in water bath at 32°C, and melatonin added as treatments: control; 100pM; 100nM; 100µM and 1mM melatonin. Then, the samples were cooled in a cold chamber at 5°C for two hours, in straws of 0.5mL and sealed. They were stored under the liquid nitrogen vapor for 15 minutes to 8cm of liquid blade and frozen with liquid nitrogen. Samples were analyzed for sperm motility, membrane integrity, acrosomal membrane, mitochondrial activity, oxidative stress and quantification of the binding capacity. The variables were subjected to analysis of variance and the means were compared by Tukey test at 5% probability. The total and progressive motility of thawed sperm were higher in samples treated with 100pM melatonin (62.99 and 45.07%, respectively; P<0.05) when compared to other treatments. The addition of different concentrations of melatonin in semen diluted with the exception of 1mM concentration, a higher percentage of cells with intact plasma membrane, as compared with the control (P<0.05). The percentage of sperm with acrosome membrane integrity was higher in the semen with 100pM melatonin (P<0.05) than the other treatments. The high mitochondrial activity was higher in spermatozoa treated with 100pM melatonin (69.30%; P<0.05). Addition of 100nM melatonin reduced the amount of TBARS after cryopreservation (2.84, P<0.05) when compared with the other treatments. After thawing, the number of sperm which bind to the perivitelline membrane was higher in the melatonin treated with 100pM (155,73; P<0.05). Therefore, melatonin addition the semen diluted can be useful to enhance the cryopreservation of sheep semen, improving fertilization rates through artificial insemination.(AU)

In vitro fertilization of porcine oocytes is affected by spermatic coincubation time / A fertilização in vitro de ovócitos suínos é afetada pelo tempo de coincubação espermático

The aim was to study the effects of different gamete coincubation times on porcine in vitro fertilization (IVF), and to verify whether efficiency could be improved by reducing oocyte exposure time to spermatozoa during IVF. In groups of 50, a total of 508 immature cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium. The COCs were cultured for 44 hours and then inseminated with in natura semen (2,000 spermatozoa/oocyte). The sperm and oocytes were coincubated according to the following treatments (T): T1 = oocytes exposed to spermatozoa for one hour (173 oocytes), T2 = oocytes exposed to spermatozoa for two hours (170 oocytes), and T3 = oocytes exposed to spermatozoa for three hours (165 oocytes). After these coincubation periods, the oocytes were washed in fertilization medium (TALP medium) to remove spermatozoa not bound to the zona pellucida and cultured in another similar medium (containing no sperm). Eighteen to twenty hours after fertilization, the putative zygotes were stained in Hoechst-33342 to evaluate the IVF results. The penetration rate was higher (P<0.05) after two hours of coincubation time than it was for one or three hours. Furthermore, 68.60% of the ova coincubated with the spermatozoa for two hours were monospermic. The oocytes exposed to spermatozoa for one hour (T1) presented a higher (P<0.01) rate of polyspermy than those in T2 and T3. Fertilization performance (%) did not differ (P>0.05) between oocytes exposed to spermatozoa for one (T1) and three hours (T3). However, optimum (P=0.048) results were obtained after two hours of coincubation, when the rate of fertilization performance was 50.16±8.52%. The number of penetrated sperm per oocyte, as well as male pronucleus formation, did not differ (P>0.05) between the treatments evaluated. Under these assay conditions, especially in relation to the sperm concentration used, gamete coincubation for a period of two hours appears to be optimal for monospermy and fertilization performance. Thus, it is the optimal time period for obtaining a large number of pig embryos capable of normal development.(AU)

Esse estudo foi realizado para avaliar os efeitos de diferentes tempos de coincubação dos gametas sobre a fertilização in vitro (FIV) de suínos e se a eficiência dessa técnica poderia ser melhorada pela redução no período que os ovócitos são expostos aos espermatozoides durante a FIV. Um total de 508 (em grupos de 50) complexos cumulus-ovócito (COCs) imaturos foram maturados no meio NCSU-37. Os COCs foram cultivados por 40-44 horas e então inseminados com sêmen in natura (2.000 espermatozoides/ovócito). Os espermatozoides e ovócitos foram coincubados de acordo com os seguintes tratamentos (T): T1 = ovócitos expostos aos espermatozoides por uma hora (173 ovócitos); T2 = ovócitos expostos aos espermatozoides por duas horas (170 ovócitos) e T3 = ovócitos expostos aos espermatozoides por três horas (165 ovócitos). Após esses períodos de coincubação, os ovócitos foram lavados em meio de fertilização (meio TALP) para remoção dos espermazoides não ligados a zona pelúcida e cultivados em outro mesmo meio (não contendo espermatozoides). Após 18-20 horas de fertilização, os prováveis zigotos foram corados com Hoechst-33342 para avaliação dos resultados da FIV. A taxa de penetração foi maior (P<0,05) após o tempo de coincubação de duas horas em comparação a uma e três horas. Além disso, 68,60% dos ovócitos coincubados com os espermatozoides por duas horas foram monospérmicos. Os ovócitos expostos aos espermatozoides por uma hora (T1) apresentaram elevada (P<0,01) taxa de polispermia em comparação com o T2 e T3. A eficiência da fertilização (%) não diferiu (P>0,05) entre os ovócitos expostos aos espermatozoides por uma (T1) e três horas (T3). Entretanto, ótimos (P=0,048) resultados foram obtidos após duas horas de coincubação, quando a taxa da eficiência da fertilização foi 50,16 ± 8,52%. O número de espermatozoides penetrados por ovócito e a formação de pro-núcleo masculino não diferiu (P>0,05) entre os tratamentos avaliados. Sob as condições de ensaio realizadas, especialmente em relação à concentração espermática utilizada, a coincubação dos gametas por um período de duas horas parece ser ótima para as taxas de monospermia e eficiência da fertilização. Portanto, um tempo provavelmente ótimo para obter um elevado número de embriões suínos capazes de ter um desenvolvimento normal.(AU)