Aplicação de microssatélites na crioconservação da diversidade genética do cavalo lusitano

O género Equus teve origem na América do Norte e alguns exemplares migraram para a Eurásia pelo Estreito de Bering, durante a última glaciação. No fim da glaciação, todos os cavalos do continente americano extinguiram-se, mas sobreviveram nas estepes da Eurásia, na Peninsula lbérica e nas florestas da Europa Ocidental e Central. O cavalo Lusitano teve a sua origem em cavalos selvagens e domesticados da Peninsula lbérica, ocorrendo uma mistura com outros animais trazidos por eventos migratórios ocorridos no passado. Os cavalos deste gene pool contribuiram para o desenvolvimento de outras raças modernas na Europa e foram mais tarde introduzidos e dispersos pelo continente Americano, tornando-se fundadores de numerosas raças do novo mundo. A raça Lusitana é uma raça equina autóctone portuguesa, com especial relevancia económica no panorama nacional e internacional. Apesar de não ser uma raça ameaçada, alguns autores defendem que a informação genealógica disponivel (pedigrees) indica que uma utilização excessiva de um reduzido número de reprodutores machos esta a diminuir a diversidade genética da raça, tendo como consequência o aumento da consanguinidade e a diminuição do tamanho efetivo da população para cerca de metade dos valores recomendados pela FAO. No entanto, a anàlise da diversidade genética com base em 16 microssatélites (Marcadores de DNA) a um grupo de 2699 machos da raça Lusitana, nascidos entre 1985 e 2010 e inscritos como reprodutores no Livro Genealógico da raça, revelou um elevado nível de diversidade, idêntico ao encontrado na maioria das raças equinas. Dada a crescente relevância da Crioconservação, omo estratégia complementar para a conservação da diversidade genética in situ, e tendo em conta que não existe criopreservação de oocitos, embriões ou sémen, do cavalo de raça Lusitana em Banco de Genes, selecionaram-se 62 machos reprodutores (garanhões) com interesse genético para a criopreservação de sémen, quer no sentido de preservar a diversidade da raça quer no da salvaguardar em caso de calamidade; ABSTRACT: The genus Equus originated in North America and some exemplary migrated to Eurasia through the Bering Strait during the last glaciation. By the end of the last glaciation, all horses on the American continent became extinct but the genus survived in the steppes of Eurasia, in the Iberian Peninsula and on the Central and West Europe forests. The Lusitano horse breed has its origins in wild and domesticated horses of the Iberian Peninsula, where a mixture with other animals brought by migratory events in the past occurred. The horses of this gene pool contributed to the development of other modern breeds in Europe and were later introduced and spread throughout the American continent, becoming founders of numerous breeds of the New World. The Lusitano horse breed, is a Portuguese native equine breed, with special economic relevance in the national and international scene. Although not being an endangered breed, some authors argue that the available genealogical information (pedigrees) indicates that an excessive use of a limited number of stallions is decreasing the genetic diversity of the breed, resulting in the increase of inbreeding and on the decrease of the effective population size to about half of the values recommended by FAO. However, the analysis of genetic diversity based on 16 microsatellites (DNA markers) in a group of 2699 males of the Lusitano horse breed, born between 1985 and 2010 and registered as Stallions in the Studbook, revealed a high level of diversity similar to that found in the majority of equine breeds. Given the growing relevance of Cryopreservation as a complementary strategy for the conservation of genetic diversity in situ and, taking into consideration the inexistence of criopreservation for oocytes, embryos and semen, in a Gene Bank, for the Lusitano horse breed, 62 breeding males (stallions) with genetic interest for semen cryopreservation were selected in order either to preserve the diversity of the breed or as safeguard in case of calamity.

The successful double cryopreservation of rabbit (Oryctolagus cuniculus) semen in large volume using the directional freezing technique with reduced concentration of cryoprotectant

Using directional freezing, Our objective was to cryopreserve rabbit semen and achieve fertility that was equal or higher than that achieved with conventional freezing. The working hypothesis was that controlling the ice-front propagation would allow redu

Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity

Objective: To investigate effects of cryopreservation on sperm motility and DNA integrity. Design: Pre-cryopreservation and post-cryopreservation analysis of motility and DNA integrity of semen and prepared sperm samples. Setting: A hospital andrology laboratory. Patient(s): Forty men attending the Regional Fertility Centre, Belfast, Northern Ireland. Intervention(s): Each sample was divided, and an aliquot was frozen unprepared. Remaining aliquots were prepared by Percoll density centrifugation (95.0:47.5) or direct swim-up procedure and divided into aliquots to allow direct comparison of fresh and frozen semen and prepared sperm (frozen with or without the addition of seminal plasma) from the same ejaculate. Samples were frozen by static-phase vapor cooling and being plunged into liquid nitrogen. Thawing was carried out at room temperature. Main Outcome Measure(s): Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay, and motility was determined using computer-assisted semen analysis. Result(s): Sperm frozen unprepared in seminal fluid appeared more resistant to freezing damage than frozen prepared sperm. Further improvements can be achieved by selecting out the subpopulation of sperm with best motility and DNA integrity and freezing these sperm in seminal plasma, making this the optimal procedure. Conclusion(s): Freezing sperm in seminal plasma improves postthaw motility and DNA integrity.

Replacing egg yolk with soybean lecithin in the cryopreservation of stallion semen

The objective of this study was to determine whether replacing the egg yolk with soybean lecithin in the Botu-Crio (R) cryodiluent would maintain the fertility of cryopreserved stallion sperm. Two experiments were performed to evaluate cell freezability. In experiment 1, sperm from 15 stallions were frozen in Botu-Crio (R) (BC) or Botu-Crio (R) which contained 45 g/L soybean lecithin (BCLS45) in place of the egg yolk. In experiment 2, we compared different concentrations of soybean lecithin: 0, 10.0, 12.5, 15.0, 17.5 and 20.0 g/L (BC, BCLS10, BCLS12.5, BCLS17.5 and BCLS20, respectively). In experiment 1, sperm frozen in BC and BCLS45 exhibited similar (P > 0.05) percentages of total motile sperm (61% and 61%, respectively); progressively motile sperm (27% and 27%, respectively) and sperm with intact plasma membranes (IMP; 53% and 57%, respectively). Similarly, sperm frozen in BC or BC containing any concentration of soybean lecithin maintained similar (P > 0.05) percentages of total motile sperm (61-68%) and progressively motile sperm (27-31%). In the first fertility trial, we used cryopreserved semen from a single stallion was inseminated into mares. The semen from the sperm that were frozen in BC diluent resulted in a higher fertility rate (66%, 16/24) compared to the sperm that were frozen in BCLS45 diluent (17%, 5/29; P < 0.01). Similarly, in a second fertility trial, the mares that were inseminated with the sperm that were frozen in BC diluent exhibited a higher fertility rate (66%, 16/24) compared to the mares that were inseminated with the sperm that were frozen in BCLS20 (40%, 10/25; Pc 0.05). Finally, in a third trial, the sperm that were frozen in BC resulted in a higher fertility rate in mares (75%, 18/24) compared to the sperm that were frozen in BCLS10 (41%, 10/24; P < 0.05). Although replacing the egg yolk in the BC cryodiluent with soybean lecithin provided similar laboratory results for stallion sperm, after cryopreservation, the sperm that was frozen with soybean lecithin in the diluent correlated with lower fertility rates. Based on these results, we concluded that the use of BCLS can be used as an alternative diluent for cryopreserving stallion sperm. However, the resulting reduced fertility rate is a matter of concern. Further studies are necessary to clarify the reasons for this decrease in fertility and to determine the optimal lecithin concentration for diluents to freeze stallion sperm. (C) 2011 Elsevier B.V. All rights reserved.

Cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa in a chemically defined extender

Aim: To establish a method for cynomolgus monkey sperm cryopreservation in a chemically defined extender. Methods: Semen samples were collected by electro-ejaculation from four sexually mature male cynomolgus monkeys. The spermatozoa were frozen in straws by liquid nitrogen vapor using egg-yolk-free Tes-Tris (mTTE) synthetic extender and glycerol as cryoprotectant. The effects of glycerol concentration (1%,3%, 5%, 10% and 15% [v/v]) and its equilibration time (10 min, 30 min, 60 min and 90 min) on post-thaw spermatozoa were examined by sperm motility and sperm head membrane integrity. Results: The post-thaw motility and head membrane integrity of spermatozoa were significantly higher (P < 0.05) for 5% glycerol (42.95 +/- 2.55 and 50.39 +/- 2.42, respectively) than those of the other groups (1%: 19.19 +/- 3.22 and 24.84 +/- 3.64; 3%: 34.23 +/- 3.43 and 41.37 +/- 3.42; 10%: 15.68 +/- 2.36 and 21.39 +/- 3.14; 15%: 7.47 +/- 1.44 and 12.90 +/- 2.18). The parameters for 30 min equilibration (42.95 2.55 and 50.39 2.42) were better (P < 0.05) than those of the other groups (10 min: 31.33 +/- 3.06 and 38. 98 +/- 3.31; 60 min: 32.49 +/- 3.86 and 40.01 +/- 4.18; 90 min: 31.16 +/- 3.66 and 38.30 +/- 3.78). Five percent glycerol and 30 min equilibration yielded the highest post-thaw sperm motility and head membrane integrity. Conclusion: Cynomolgus monkey spermatozoa can be successfully cryopreserved in a chemically defined extender, which is related to the concentration and the equilibration time of glycerol.

Cryopreservation of flounder (Paralichthys olivaceus) sperm with a practical methodology

A simple and convenient protocol for the cryopreservation of the flounder (Paralichthys olivaceus) sperm was established for "on the spot" cryopreservation of large quantities of semen. The use of three cryoprotectants, dimethyl sulphoxide (DMSO), glycerol (Gly) and methanol was tested in the method. The percentage of motile sperm present in semen after it had been frozen and thawed in the presence of DMSO, Gly or methanol was 60.5 +/- 3.6, 79.17 +/- 4.5 and 13.25 +/- 4.7%, respectively. The fertilization rates of this sperm were 67.06 +/- 15.1, 76.20 +/- 10.0 and 44.93 +/- 22.6%, while the hatching rates of eggs fertilized with this sperm were 37.40 +/- 8.3, 48.18 +/- 25.7 and 23.35 +/- 10.8%, respectively. It was found that Gly and DMSO were better cryoprotectants than methanol, with Gly giving the best overall results. Under scanning electron microscopy, it could be seen that while the majority of the frozen-thawed sperm remained morphologically normal, some exhibited lost or dilated mitochondria, swollen mid-pieces, broken tails, or damaged cell membrane, which probably caused the decrease in motility and fertility of the frozen-thawed sperm. (C) 2003 Elsevier Science Inc. All rights reserved.

Assessment of DNA integrity and morphology of ejaculated sperm from fertile and infertile men before and after cryopreservation

Cryopreservation of human spermatozoa is extensively used in artifical insemination and IVF programmes. Despite various advances in cryopreservation methodology, the recovery rate of functional post thaw spermatozoa remains mediocre, with sperm motility being significantly decreased after freezing. The aim of this study was to investigate the effects of cryopreservation on both DNA integrity and morphology of spermatozoa from fertile and infertile men. Semen samples were obtained from 17 fertile men and 40 infertile men. All samples were prepared by discontinuous Percoll density centrifugation ( 95.0:47.5). Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. Aliquots for cryopreservation were mixed with a commercial cryoprotectant and frozen by static phase vapour cooling before plunging into liquid nitrogen. Thawing was carried out slowly at room temperature. Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis ( comet ) assay and sperm morphology analysed using the Tygerberg criteria. DNA of semen and prepared sperm from fertile men was found to be unaffected by cryopreservation. In marked contrast, spermatozoa from infertile men were significantly damaged by freeze- thawing. Cryopreservation had a detrimental effect on morphology of semen and prepared sperm from fertile and infertile men.

Spermatozoon ultrastructure and sperm cryopreservation of the Brazilian dry season spawner fish pirapitinga, Brycon nattereri

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cryopreservation effects on domestic cat epididymal versus electroejaculated spermatozoa

Frozen-thawed epididymal spermatozoa have already been successfully used in artificial insemination in the domestic cat, proving to be a valuable resource for the reproduction of felid species, which are threatened with extinction. The aim of this study was to compare the effects of freezing and thawing on domestic cat semen collected by electroejaculation (EL) and from the epididymides (EP) and vasa deferentia. Ten adult cats were anesthetized, electroejaculated and immediately thereafter, orchiectomized. Epididymal spermatozoa were collected through the compression of caudae epididymidis and vasa deferentia. Spermatozoa were frozen-thawed following a single protocol. Sperm motility, sperm progressive status (0-5), plasma membrane integrity and morphology (light and transmission electron microscope) were assessed on two occasions, immediately after collection and after freezing and thawing. There were no significant differences between the electroejaculated and epididymal fresh or frozen-thawed spermatozoa for any of the variables. However, the incidence of acrosome defects after freezing and thawing increased by 19% based on light microscopy, whereas ultrastructural images revealed acrosome damages in most sperm cells. Since these acrosomal changes are known to affect sperm fertilising capacity, further studies are needed to optimize cryopreservation techniques for epididymal as well as electroejaculated domestic cat spermatozoa. (c) 2006 Elsevier B.V. All rights reserved.

Comparison of in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or new lecithin based extenders

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