Reproductive characteristics of stallions during the breeding and non-breeding season in a tropical region

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of a Low Dose of Equine Purified FSH to Induce Multiple Ovulations in Mares

The effects of a low dose of equine purified FSH (eFSH) on incidence of multiple ovulations and embryo recovery rate in mares were studied. During the physiological breeding season in Brazil (19 degrees 45'45'S), 14 Mangalarga Marchador donor mares were used in a crossover study and another 25 mares of the same breed, between 3 years and 12 years of age were used as recipients for the embryo transfers. Donors were monitored during two consecutive oestrus cycles, an untreated control cycle followed by a treated cycle, when eFSH was administered. In both cycles, after an embryo collection attempt on day 8 post-ovulation all mares received 7.5 mg dinoprost and had their two largest follicles tracked daily by ultrasonography until the period of ovulation. Mares were inseminated every 48 h with extended fresh semen from a single stallion after the identification of a 35-mm follicle until the period of ovulation. Ovulations were induced by intravenous administration of 2.500 IU of human chorionic gonadotropin, upon detection of a 35- to 40-mm follicle. In the treated cycle, 5 mg eFSH was given intramuscularly once a day, from day 8 post previous ovulation until at least one follicle reached 35 mm in diameter. Embryo flushes were performed on day 8 of dioestrus (day 0 = ovulation). Treatment with eFSH resulted in higher (p < 0.05) ovulation rate and incidence of multiple ovulations compared to the control (1.6 vs 1.0 and 50% vs 0%, respectively - one mare had triple ovulation). However, embryo recovery rates in the control and treated cycles were similar (0.8 and 1.0, respectively; p > 0.05). Pregnancy rates in the recipient mares following embryo transfer were similar for the control and eFSH cycles (11/11 and 10/14, respectively). Additional studies are necessary in order to develop a low-dose protocol for the use of eFSH that brings a more consistent contribution to the efficiency of commercial equine embryo transfer programs.

Freezing of stallion epididymal sperm

Inseminations with frozen-thawed epididymal sperm have resulted in low-pregnancy rates of mares. If fertility of epididymal sperm could be improved, it would help to preserve genetic material from stallions that have suffered severe injuries, been castrated or have died. The aim of the present study was to investigate the effect of different extenders and pre-freezing addition of capacitation media on freezability of epididymal sperm and on storage at 5 degrees C for 24 h. In experiment 1, epididymal sperm samples were diluted and subsequently frozen with three different extenders: Botu-Crio((R)), EDTA-Lactose and INRA-82. Motility analysis using computer assisted sperm analyzer (CASA) demonstrated better motility for sperm in Botu-Crio((R)) than in the other extenders; EDTA-Lactose yielded better motility than INRA-82 on most evaluated parameters. There was no difference in membrane integrity among the studied extenders. From 18 inseminated mares, 12 (66%) were pregnant 15 days after AI with frozen-thawed epididymal sperm showing that Botu-Crio((R)) was able to maintain the fertility potential. In experiment 2, the effect of incubation of epididymal sperm before freezing in three capacitation media (Fert Talp, Sperm Talp, Talp + Progesterone), seminal plasma, or control was tested. Based on post-thaw motility evaluation by CASA, samples incubated in Sperm Talp showed better motility values. There were no differences in plasma or acrosomal membranes or in mitochondrial potential among groups. We concluded that Botu-Crio((R)) was better than the other extenders in the ability to preserve epididymal sperm and that pre-freeze addition of Sperm Talp was also beneficial. (c) 2008 Published by Elsevier B.V.

Effect of Progesterone and Ionomycin on Domestic Cat Sperm Motility Patterns and Acrosome Reaction

This study was aimed at assessing the changes in sperm motion patterns and the percentage of acrosome reaction (AR) in domestic cat semen after treatment with either ionomycin or progesterone (P(4)). Ten ejaculates were collected from five tomcats using an artificial vagina, and were diluted, centrifuged and resuspended in a capacitation medium. Samples were evaluated and divided into seven equal aliquots and, after 2 h at 25 degrees C, were incubated for 30 min at 38 degrees C in 5% CO(2) and then analyzed. Computer-assisted sperm analysis and a combination of three fluorescent probes were used to assess sperm plasma, acrosomal membrane integrity and mitochondrial transmembrane potential. Thirty minutes after the start of incubation, P(4) was added (10 mu g/ml) to the P1 group. Groups P2 and P3 were supplemented with P(4) (10 and 20 mu g/ml, respectively) only after 2 h of incubation, and groups I1 and I2 were supplemented with ionomycin (4 and 8 mu M, respectively) 2 h after incubation. Group E was supplemented with ethanol (0.6%) at 2 h after incubation and group C received no supplementation. Ionomycin and P(4) treatments led to a hyperactivation-like sperm motion and an increase (p < 0.05) in the percentage of AR. Although a higher (p < 0.05) percentage of AR was obtained in group I2 when compared with all P(4) groups, a decrease (p < 0.05) in total and progressive motility was observed in I2 group. As I1 group was similar to I2 to induce AR without diminishing sperm motility, we can conclude that ionomycin at 4 mu M seems to be more suitable to trigger AR in domestic cat sperm.

Motility and viability of ram sperm cryopreserved in a Tris-egg yolk extender supplemented with anti-oxidants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Embryo Transfer Efficiency of Quarter Horse Athletic Mares

This study was designed to compare embryo recovery rates and pregnancy rates of athletic and breeding Quarter Horse mares in a tropical warm climate. Thirty-nine barrel racing mares in training and 135 breeding mares as control donors were included. During the training period, the ambient temperature ranged from 31 degrees C to 36 degrees C and the average humidity from 70% to 90%. After the detection of a 35-mm follicle by ultrasound, ovulation was induced with 1 mg of deslorelin acetate (i.m), and insemination was performed 24 hours later with cooled and fresh semen from different fertile stallions. Embryos were collected on day 8 postovulation. The body temperature (rectal) was evaluated from eight athletic donor mares randomly selected from the same studied group. A total of 138 and 657 embryo collections were carried out on training and breeding mares, respectively, with a total of 105 (76%) and 466 (71%) embryos collected (P > .05). Similarly, no differences (P > .05) were observed for the pregnancy rates on day 15 (82/105, 78% vs. 370/466,79%), and day 40 (73/105, 69% vs. 328/466,70%) between the training and breeding donor mares. Just after training, the body temperature increased to an average of 39.4 degrees C and the respiratory rate from 14.5 to 35.3 breaths per minute. The results of the present study showed that embryo production from appropriately trained donor mares in good condition were similar to non-athletic broodmares. (C) 2011 Published by Elsevier B.V.

Advances in stallion's epididymal sperm technology

Freezing epididymal sperm is a method to preserve germplasm from animals with not only high genetic potential but also endangered species. In the equine some owners have chosen this possibility in cases of either severe illness or death of stallions. However, the lack of knowledge and poor published results of such technique hampers its propagation. New procedures have allowed some improvement on fertility rates of frozen sperm from the epididymis of stallions. The aim of this study is to report the advances on processing and cryopreservation of samples from the stallion's epididymal semen.

PCR Multiplex fluorescente para detecção de bactérias em sêmen bovino

Este estudo teve como objetivo avaliar o limiar de detecção da técnica de PCR multiplex fluorescente aliada a eletroforese capilar na detecção de agentes infecciosos em amostras de sêmen experimentalmente contaminadas com concentrações decrescentes das bactérias Brucella abortus, Leptospira interrogans sorovar pomona, Campylobacter fetus e Haemophilus somnus. Amostras de sêmen bovino foram experimentalmente contaminadas com concentrações decrescentes de bactérias obtidas através de diluições seriadas na base 10 de modo a obter-se amostras contendo desde 1 vez até 10-7 bactérias/mL a partir da concentração inicial de Leptospira pomona, Brucella abortus, Campylobacter fetus e Haemophilus somnus. As diluições foram efetuadas individualmente para cada bactéria, bem como nas diferentes concentrações necessárias para a padronização do teste de multiplex PCR. As extrações de DNA de todas as soluções contendo espermatozóides e bactérias analisadas no presente estudo foram realizadas segundo protocolo descrito por Heinemann et al. (2000). Os produtos de PCR multiplex foram avaliados por eletroforese em gel de poliacrilamida 8% e separação eletroforética por sistema capilar em equipamento automático de análise de fragmentos de DNA MegaBace. Observou-se a amplificação de fragmentos de 193pb, 330pb, 400pb e 415pb a partir do DNA de B. abortus, L. pomona, H. somnus, C. fetus, respectivamente. Na análise por eletroforese capilar de produtos da PCR multiplex do DNA para detecção simultânea dos quatro patógenos observou-se a sinal de positividade até a diluição de 10-3 bactérias/mL vezes da concentração inicial da solução estoque de cada bactéria. A técnica de PCR multiplex aliada à eletroforese capilar foi usada pela primeira vez para o diagnóstico direto de quatro bactérias patogênicas no sêmen, demonstrando ser um método rápido na detecção de bactérias causadoras de doenças reprodutivas.

Qualidade espermática de sêmen de cães naturalmente infectados por Leishmania sp:

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Copulatory efficiency and fertility in male rats exposed perinatally to flutamide

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Heparin-binding proteins of seminal plasma in Nellore bulls

O objetivo deste estudo foi identificar proteínas ligadoras à heparina no plasma seminal de touros Nelore (Bos taurus indicus). Para tanto, foram selecionados quatro touros entre 30 e 36 meses de idade e peso aproximado de 500-550kg. Após centrifugação, amostras do plasma seminal foram misturadas e as proteínas ligadoras à heparina foram isoladas por meio da cromatografia por afinidade. As frações após a eluição foram agrupadas para caracterização das bandas protéicas (SDSPAGE, 12,5%). Foram identificadas oito bandas protéicas variando entre 15 e 63kDa. Duas proteínas com 22 e 25kDa foram similares às descritas em touros Bos taurus taurus. Outras proteínas identificadas com 39, 53, 58 e 63kDa ainda não foram descritas e possivelmente sejam específicas para Bos taurus indicus.

Virus present in the reproductive tract of asymptomatic drones of honey bee (Apis mellifera l.), and possible infection of queen during mating

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Is The amphibian tree of life really fatally flawed?

Wiens (2007, Q. Rev. Biol. 82, 55-56) recently published a severe critique of Frost et al.'s (2006, Bull. Am. Mus. Nat. Hist. 297, 1-370) monographic study of amphibian systematics, concluding that it is a disaster and recommending that readers simply ignore this study. Beyond the hyperbole, Wiens raised four general objections that he regarded as fatal flaws: (1) the sampling design was insufficient for the generic changes made and taxonomic changes were made without including all type species; (2) the nuclear gene most commonly used in amphibian phylogenetics, RAG-1, was not included, nor were the morphological characters that had justified the older taxonomy; (3) the analytical method employed is questionable because equally weighted parsimony assumes that all characters are evolving at equal rates; and (4) the results were at times clearly erroneous, as evidenced by the inferred non-monophyly of marsupial frogs. In this paper we respond to these criticisms. In brief: (1) the study of Frost et al. did not exist in a vacuum and we discussed our evidence and evidence previously obtained by others that documented the non-monophyletic taxa that we corrected. Beyond that, we agree that all type species should ideally be included, but inclusion of all potentially relevant type species is not feasible in a study of the magnitude of Frost et al. and we contend that this should not prevent progress in the formulation of phylogenetic hypotheses or their application outside of systematics. (2) Rhodopsin, a gene included by Frost et al. is the nuclear gene that is most commonly used in amphibian systematics, not RAG-1. Regardless, ignoring a study because of the absence of a single locus strikes us as unsound practice. With respect to previously hypothesized morphological synapomorphies, Frost et al. provided a lengthy review of the published evidence for all groups, and this was used to inform taxonomic decisions. We noted that confirming and reconciling all morphological transformation series published among previous studies needed to be done, and we included evidence from the only published data set at that time to explicitly code morphological characters (including a number of traditionally applied synapomorphies from adult morphology) across the bulk of the diversity of amphibians (Haas, 2003, Cladistics 19, 23-90). Moreover, the phylogenetic results of the Frost et al. study were largely consistent with previous morphological and molecular studies and where they differed, this was discussed with reference to the weight of evidence. (3) The claim that equally weighted parsimony assumes that all characters are evolving at equal rates has been shown to be false in both analytical and simulation studies. (4) The claimed strong support for marsupial frog monophyly is questionable. Several studies have also found marsupial frogs to be non-monophyletic. Wiens et al. (2005, Syst. Biol. 54, 719-748) recovered marsupial frogs as monophyletic, but that result was strongly supported only by Bayesian clade confidence values (which are known to overestimate support) and bootstrap support in his parsimony analysis was < 50%. Further, in a more recent parsimony analysis of an expanded data set that included RAG-1 and the three traditional morphological synapomorphies of marsupial frogs, Wiens et al. (2006, Am. Nat. 168, 579-596) also found them to be non-monophyletic.Although we attempted to apply the rule of monophyly to the naming of taxonomic groups, our phylogenetic results are largely consistent with conventional views even if not wth the taxonomy current at the time of our writing. Most of our taxonomic changes addressed examples of non-monophyly that had previously been known or suspected (e.g., the non-monophyly of traditional Hyperoliidae, Microhylidae, Hemiphractinae, Leptodactylidae, Phrynobatrachus, Ranidae, Rana, Bufo; and the placement of Brachycephalus within Eleutherodactylus, and Lineatriton within Pseudoeurycea), and it is troubling that Wiens and others, as evidenced by recent publications, continue to perpetuate recognition of non-monophyletic taxonomic groups that so profoundly misrepresent what is known about amphibian phylogeny. (C) The Willi Hennig Society 2007.

Seminal analysis, cryogenic preservation, and fertility in matrinxã fish, Brycon cephalus (Günther, 1869)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Detection of Toxoplasma gondii in the reproductive system of male goats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)