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

Data_Sheet_1_Ability of donkey sperm to tolerate cooling: Effect of extender base and removal of seminal plasma on sperm parameters and fertility rates in mares.docx

Artificial insemination using cooled-transported semen has marked importance in equine breeding programs around the world, and the high value of mules has generated avid interest in donkey semen biotechnology. However, donkey semen cools poorly in commercially available equine extenders. Therefore, this study aimed to develop approaches to improve the ability of donkey semen to tolerate cooling. Ejaculates of seven donkeys (n = 21) were cooled at 5°C for 48 h in three different extenders (milk-based, SM; sodium caseinate-based, SC; or egg yolk-based, EY) in the presence or absence of seminal plasma (centrifugation, C). Sperm motility, plasma membrane integrity (PMI), plasma membrane stability (PMS), mitochondrial membrane potential (HMMP), intracellular hydrogen peroxide (H2O2), and intracellular superoxide (O2−) were assessed before, 24 h, and 48 h post-cooling. In addition, 15 mares (163 estrous cycles) were randomly inseminated with semen from two jacks (Jack 1, n = 90; Jack 2, n = 73) previously cooled for 24 h under one of the treatments (SM, SC, EY, SM-C, SC-C, or EY-C). Groups EY, SC-C, and EY-C (P < 0.05) demonstrated superior sperm analytical parameters to SM at 24 and 48 h. Centrifugation positively affected sperm analytical parameters in cooled donkey semen extended in SM and SC (P < 0.05). Mares bred with semen extended in SC (67%, 18/27), SC-C (89%, 24/27), EY (89%, 25/28), or EY-C (74%, 20/27) had significantly greater conception rates than mares bred with SM (33%, 9/27; P < 0.05). Mares bred with SM-C had intermediate conception rates (59%, 16/27). In conclusion, SC and EY improved the cooling ability and fertility of donkey semen in horse mares, and centrifugation positively affected donkey semen extended in SM.</p

Cooling of ejaculated and epididymal stallion sperm

After a serious injury or sudden death, epididymis cauda sperm recovery and cryopreservation may present as the last opportunity to obtain genetic material from a valuable stallion. This study evaluated the viability of cooled equine sperm collected by three different methods: sperm of ejaculated (G1), sperm recovered from the epididymal cauda immediately after orchiectomy (G2) and sperm recovered from the epididymal cauda after storage for 24 hours at 5°C (G3). To obtain G1 sperm, two ejaculates were collected. After 1 week, all stallions underwent a bilateral orchiectomy, and one of the removed epididymides was flushed to obtain G2 sperm. The contralateral epididymis was stored at 5°C for 24 hours before being flushed to obtain G3 sperm. The sperm samples were evaluated immediately after the addition of the refrigeration extender, and after 24 and 48 hours of storage at 5°C. After 24 and 48 hours of storage, the epididymal sperm demonstrated higher motility traits when compared to the ejaculated sperm (P<0.05). These results indicate that sperm recovered from the epididymal cauda of stallions are more resistant to the cooling process, with higher kinetic parameters and plasma membrane integrity when compared to the ejaculated sperm.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

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

One of the main factors known to influence quality and fertility of bovine cryopreserved semen is the extender used. In this matter, a great worldwide interest has been directed to the development of chemically defined media, free of animal origin products. The objective of the present study was to compare the efficacy of three bovine semen extenders: Iris-fructose (IRIS, control with 20% egg yolk), Botu-Bov (R) (BB; 20% egg yolk), and Botu-Bov (R)-soy lecithin (BB-L; 1% soy lecithin). Towards this aim, post-thaw computer assisted sperm analysis (CASA), sperm membrane and acrosome integrity were evaluated (Experiment 1). Additionally, cryopreserved samples were used in a fixed time artificial insemination program aiming to evaluate in vivo fertility (pregnancy per insemination-P/AI; Experiment 2). Despite the higher straightness and linearity found for samples cryopreserved in BB and BB-L when compared to those cryopreserved in IRIS, egg yolk based extenders provided higher total and progressive motilities, percentage of rapid sperms and intact membrane cells (P < 0.05). Furthermore, P/IA was higher in samples cryopreserved in egg yolk based extenders when compared to soy lecithin [TRIS=59.26(a) (64/108), BB=62.37(a) (58/93), and BB-L=36.45(b) (35/96)]. Although soy lecithin represents an alternative for the development of chemically defined extenders with decreased risk of biological contamination, egg yolk based extenders are more efficient on the preservation of bovine post-thaw sperm viability and fertility. (C) 2012 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP