Amides as cryoprotectants for freezing stallion semen: A review

Stallion semen cryopreservation, despite its impact on the horse industry, is not an established technology. During the last years, a number of modifications have been proposed to the freezing process, however, a large population of stallions still have poor semen quality and fertility after frozen-thawed. Glycerol toxicity could be a reason for the variation on stallion sperm freezability. There are limited publications concerning the use of alternative cryoprotectants for equine sperm. Glycerol is contraceptive for some species and other cryoprotectors, such as amides, have been show to be a good option for freezing semen of these species. Recent reports have shown encouraging data respecting the use of amides as cryoprotectants for stallions, with more remarkable improvements for semen from stallions that freeze poorly when glycerol is used. (C) 2005 Elsevier B.V. All rights reserved.

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.

Perfil em SDS-PAGE das proteínas do plasma seminal e sua relação com a congelabilidade do sêmen de touros doadores da raça gir

Foram utilizados dez animais doadores de sêmen em nível de Central de Inseminação Artificial, da raça Gir, divididos em dois grupos, de acordo com o grau de congelabilidade do sêmen de cada animal. Os animais com sêmen de alta congelabilidade foram aqueles cuja porcentagem de ejaculados viáveis pós-descongelação foi superior a 80%. O grupo de baixa congelabilidade tinha animais com porcentagem menor que 50% de ejaculados viáveis pós-descongelação. Os critérios de avaliação da viabilidade do sêmen e seleção dos animais foram definidos pelo controle de qualidade do Departamento de Produção da Central de Inseminação Artificial. Foram feitas quatro coletas semanais consecutivas, sendo que obtiveram-se as amostras de plasma seminal por centrifugação a 1.500 g por 15 a 20 minutos a 4°C, momentos após a coleta do sêmen em vagina artificial. O plasma seminal foi dialisado em membrana de celulose, em tampão Tris-Glicina pH-7,4 por 24 horas a 4°C, em agitação lenta e constante. As amostras foram padronizadas em 1,0 mg/ml de proteína total, por diluição em tampão Tris-HCl 62mM pH-6,2 mais 20% de glicerol e 4% de SDS. Através de eletroforese do tipo SDS-PAGE, foram feitas as corridas em gel a 13%. A corrida foi feita com a constante de 25 mA, por um período de 5 horas. A coloração do gel foi feita por Coomassie Brilliant Blue. Pelos resultados obtidos, verificou-se que existe uma banda no grupo de alta congelabilidade, cujo fragmento polipeptídico desta proteína tem Mr (mobilidade relativa) 20,3 e PM (peso molecular) aproximado de 61.800 Da. Esta banda não foi detectada nas amostras do grupo de baixa congelabilidade, o que sugere ser um possível marcador bioquímico quanto ao potencial de criopreservação do sêmen de bovinos.

Proteômica para investigar a congelabilidade do semen bovino

The present study was undertaken the protein composition in 2D-electrophoretic pattern (2DE) of the seminal plasma (SP) can interfere in the semen bull freezability, and if we can use that for predicting semen bull freezability. Samples were obtained of 20 bulls (different breeds) with a minimum of 3 years history semen production in commercial semen collection Center. All animals ranged between 2 - 7 years of age. The semen freezability was calculated by # of thawed and approved ejaculates / # of ejaculates submitted to cryopreservation (after semen evaluation and approved to submitted to freeze). The bulls were divided in 3 groups: HIGH (=>80% ejaculates approved); MEDIUM (>60% and <79% ejaculates approved); LOW (=<59% ejaculates approved); the pattern and criteria were the same used in the routine of the commercial semen Center. 68 gels were carried through by 2DE of SP samples indicated 225 detected spots with protein different amount (VION) comparing. Comparing bull ́s semen freezability and VION of each spot found difference among 2 spots from High and Low, even considering just spots with % of detection frequency bigger than 75%. The taurine bulls demonstrated more homogeneous profile when comparing with zebu bulls, considering number and frequency of appearance of spots. The results showed that proteomics can be a useful tool to predict the semen freezability, but we ́ll need to study better the interactions between sperm membrane, seminal plasma and extender to comprehend better which proteome phenotype interfere positive or negatively in the semen freezability.

Effects of Pentoxifylline on Equine Epididymal Sperm

This study evaluated whether pentoxifylline (PTX) present in the flushing extender influenced the function of equine epididymal spermatozoa after recovery and after thawing. For this experiment, 58 testicles from 29 Brazilian Jumping Horses were used. Cauda epididymides of each stallion were separated and flushed with a skim milk extender, with or without 7.18 mM PTX and then subjected to the freezing process. Samples flushed with the extender containing PTX showed a significant increase in total motility, progressive motility, straight line velocity, curvilinear velocity, and percentage of rapid sperm immediately after the recovery of epididymal sperm and after 15 minutes of incubation at 37°C (P < .05). However, the presence of PTX in the flushing extender did not affect the post-thaw motility parameters or plasma membrane integrity (P > .05). The results of this study showed that the PTX present in the flushing extender improved motility parameters of recently recovered epididymal sperm and had no deleterious effects on plasma membrane integrity and freezability of equine epididymal sperm. © 2013 Elsevier Inc. All rights reserved.

Efeito do meio e da dose inseminação sobre a congelabilidade e fertilidade de espermatozóides recuperados da cauda do epidídimo de garanhões

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

Estudo de diferentes sistemas e curvas de congelação na eficiência da congelabilidade e fertilidade de sêmen equino

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efeito do meio diluidor e da dose inseminante sobre a congebilidade e fertilidade do sêmen bovino utilizado em programas de inseminação artificial em tempo-fixo(LATF)

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

Inovações metodológicas na congelação de sêmen bovino

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

Efeito da substituição da gema de ovo pela lecitina de soja na criopreservação de sêmen equino

Pós-graduação em Medicina Veterinária - FMVZ

Influência do sêmen homospérmico, heterospérmico e do plasma seminal heterólogo sobre os parâmetros espermáticos e produção de embriões bovinos in vitro

Pós-graduação em Medicina Veterinária - FMVZ

Resistência omótica, congelabilidade e fertilidade do sêmen de garanhões frente a diferentes crioprotetores

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

Criopreservação e fertilidade de espermatozoides do ejaculado e recuperados da cauda do epidídimo de garanhões subférteis

Pós-graduação em Medicina Veterinária - FMVZ

Características físicas e morfológicas do sêmen de Bos taurus e Bos indicus antes e após a criopreservação

Pós-graduação em Medicina Veterinária - FCAV

Effect of Multiple Freezing of Stallion Semen on Sperm Quality and Fertility

To increase the efficiency of equine semen, it could be useful to split the artificial insemination dose and refreeze the redundant spermatozoa. In experiment I, semen of 10 sires of the Hanoverian breed, with poor and good semen freezability, was collected by artificial vagina, centrifuged, extended in INRA82 at 400 × 106 sperm/mL, and automatically frozen. After this first routinely applied freezing program, semen from each stallion was thawed, resuspended in INRA82 at 40 × 106 sperm/mL, filled in 0.5-mL straws, and refrozen. These steps were repeated, and sperm concentration was adjusted to 20 × 106 sperm/mL after a third freezing cycle. Regardless of stallion freezability group, sperm motility and sperm membrane integrity (FITC/PNA-Syto-PI-stain) decreased stepwise after first, second, and third freezing (62.3% ± 9.35, 24.0% ± 15.4, 3.3% ± 4.34, P ≤ .05; 29.6% ± 8.64, 14.9% ± 6.38, 8.3% ± 3.24, P ≤ .05), whereas the percentage of acrosome-reacted cells increased (19.5% ± 7.59, 23.9% ± 8.51, 29.2% ± 6.58, P ≤ .05). Sperm chromatin integrity was unaffected after multiple freeze/thaw cycles (DFI value: 18.6% ± 6.6, 17.2% ± 6.84, 17.1% ± 7.21, P > .05). In experiment II estrous, Hanoverian warmblood mares were inseminated with a total of 200 × 106 spermatozoa of two stallions with either good or poor semen freezability originating from the first, second, and third freeze/thaw cycle. First-cycle pregnancy rates were 4/10, 40%; 1/10, 10%; and 0/10, 0%. In conclusion, as expected, sperm viability of stallion spermatozoa significantly decreases as a consequence of multiple freezing. However, sperm chromatin integrity was not affected. Pregnancy rates after insemination of mares with refrozen semen are reduced.