Comparison of Apoptotic Cells Between Cryopreserved Ejaculated Sperm and Epididymal Sperm in Stallions

AbstractThe development of a reliable technique to freeze epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The aim of this study was to compare the apoptotic indices of sperm obtained from ejaculate, sperm recently recovered from the epididymides (EP), and sperm recovered from epididymides stored at 5°C for 24 hours (EP-stored). For the first category, two ejaculates from seven stallions were collected and then submitted to cryopreservation using an egg yolk-based extender. One week after the last semen collection, the stallions were submitted to bilateral orchiectomy, and sperm from one of the cauda epididymis was harvested immediately after castration (EP). The remaining testicle was stored in a passive refrigeration container at 5°C for 24 hours before the cauda epididymal sperm was harvested (EP-stored). Sperm harvesting from the epididymis for EP and EP-stored was performed by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender. The recovered sperm was then cryopreserved using the egg yolk-based extender. Sperm motility parameters were studied by computer-assisted semen analysis, and apoptosis was estimated by measuring caspase activity and membrane phospholipid translocation using epifluorescence microscopy. The samples were evaluated immediately (0 hour) and 8 hours after thawing. At 0 hour, no differences in sperm parameters were observed among the groups, but after 8 hours, significant statistical differences were observed in sperm motility parameters and plasma membrane integrity among the treatment groups. In addition, viable cells with no apoptotic signs were more prevalent in EP and EP-stored, suggesting that epididymal sperm is less sensitive to the cold shock caused by sperm cryopreservation

Different extenders in the cryopreservation of bovine epididymal spermatozoa

The objective of this study was to evaluate the effects of two different egg yolk extenders incubated with or without Sperm Talp on the motility and plasma membrane integrity of cryopreserved bovine epididymal spermatozoa after freezing. Twenty-five testicles with epididymides from mature bulls were collected at the abattoir. Epididymal sperm recovery was performed by retrograde flushing using a skim milk-extender (Botu-Semen™). After recovery, sperm were incubated either without or with Sperm Talp and then submitted to centrifugation. For the freezing process, half of the testes were processed with Tris egg yolk extender, and half were processed with Botu-Bov™ egg yolk extender. Samples incubated in Sperm Talp exhibited better results than epididymal spermatozoa that were incubated without Sperm Talp (p<0.05). Both Botu-Bov™ and Tris could be utilised to freeze sperm from the bovine epididymides if the sperm were previously incubated with Sperm Talp. The extenders examined in this work did not differ in their effect on plasma membrane integrity after freezing.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

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

Effect of glycerol on the viability and fertility of cooled bovine semen

The aim of the present study was to compare the viability and fertility of bovine semen diluted in Botu-Bov (BB) commercial extender with and without the cryoprotectant glycerol then cooled at 5 C for 24 hours in the Botu-Flex passive cooling system and of semen diluted in BB with glycerol then frozen. One ejaculate of 30 Nelore Bos taurus indicus bulls between 24 and 30 months of age was used for in vitro analysis. Sperm kinetics and cell viability were analyzed using computer-assisted sperm analysis and flow cytometry, respectively. Three Nelore bulls approximately 30 month old were used for in vivo test using fixed-time artificial insemination for the fertility analysis. The ejaculates were divided into three experimental groups: semen in BB extender with 7% glycerol cooled at 5 degrees C for 24 hours (cooled semen with cryoprotectant), semen in BB without glycerol cooled at 5 degrees C for 24 hours (cooled semen without cryoprotectant), and semen diluted in BB with 7% glycerol then subsequently frozen rather than cooled (frozen semen). For the fertility analysis, 762 Nelore cows (B taurus indicus) were randomly inseminated using fixed-time artificial insemination. For the groups corresponding to cooled semen with cryoprotectant, cooled semen without cryoprotectant, and frozen semen, 278, 268, and 216 cows were inseminated, respectively, and the resulting conception rates were 51% a, 44%ab and 41%b (P < 0.05), respectively. In conclusion, the fertility rates improved, when samples were cooled with glycerol at 5 degrees C for 24 hours compared with the frozen samples. (C) 2015 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP