The influence of Percoll (R) density gradient centrifugation before cryopreservation on the quality of frozen wisent (Bison bonasus) epididymal spermatozoa

Background: The wisent (Bison bonasus) is a species that has undergone a population bottleneck. Homozygosity is prevalent within the population and may have a negative impact on semen quality in wisent bulls. Semen samples containing a large amount of functionally and morphologically impaired or dead spermatozoa have lower tolerance for cryopreservation process. Such samples are prone to involve damage acrosomes, to produce and release reactive oxygen which negatively affects proper function of spermatozoan. It is a good practice to select intact and viable gametes before subjecting the sample to cryopreservation to improve the efficiency of this process. The aim of this study was to assess the ability of Percoll (R) density gradient centrifugation in order to improve the quality of wisent spermatozoa after cryopreservation. Spermatozoa samples were analysed with computer-assisted semen analysis system and flow cytometry. Results: Percoll (R) density gradient centrifugation resulted in increased percentage of motile spermatozoa, higher proportion of spermatozoa with normal morphology and proper functionality but also in a significant reduction of the total number of gametes. Nevertheless, the concentration of frozen spermatoza was still sufficient for obtaining a few complete insemination doses suggested for cattle from each epididymis. Conclusions: While creating a high-quality genetic reserve, for in vitro fertilisation purposes, eliminating detritus and improving the overall quality of samples is more important than total number of spermatozoa. For these reasons, the achievement of higher post thaw quality of spermatozoa justifies the purification of samples by centrifugation in a Percoll (R) density gradient prior to the cryopreservation process

Influence of elevated temperature on bovine oviduct epithelial cells (BOECs)

<div><p>The aim of this study was to evaluate the influence of elevated temperature on bovine oviduct epithelial cells (BOECs), based on the expression and localization of both heat shock protein 70 (HSP70), responsible for the cellular defence mechanism, and oviduct specific glycoprotein 1 (OVGP1) which is the most important embryotrophic protein. BOECs were cultured alone and co-cultured with cattle embryos at control (38.5°C) and elevated temperature (41°C) for 168 h. The elevated temperature had no effect on the viability of BOECs but exerted a negative effect on embryo development. The elevated temperature increased the expression of HSP70 and decreased the expression of OVGP1 at both mRNA and protein levels in BOECs cultured alone and those co-cultured with embryos. However, the presence of embryos limited the decrease in OVGP1 expression in BOECs at elevated temperature but did not alter the expression of HSP70. These results demonstrate for the first time the influence of elevated temperature on BOECs, consequently providing insights into the interactions between the embryo and the oviduct at elevated temperature.</p></div

The expression of ovgp1 mRNA in BOECs cultured alone and co-cultured with cattle embryos at control (38.5°C) and elevated (41°C) temperatures for 168 h post fertilization.

<p>Data presented as Mean±SEM. Bars with different letters denote statistical differences at P<0.01.</p

Oligonucleotide primers used in Real Time PCR assays.

<p>Oligonucleotide primers used in Real Time PCR assays.</p

Fluorescent analysis of HSP70 in BOECs cultured alone and co-cultured with cattle embryos at control (38.5°C) and elevated (41°C) temperatures for 168 h post fertilization.

<p>A) Representative images of immunofluorescent staining of HSP70 in BOECs cultured alone at control (38.5°C) (I) and elevated (41°C) (II) temperatures, or co-cultured with cattle embryos at control (38.5°C) (III) and elevated (41°C) (IV) temperatures. Nuclei were counterstained with Hoechst 33342 (blue); HSP70 localization was determined using antibody against HSP70 and Alexa Fluor 488-conjugated secondary antibody (green). B) The IOD values of HSP70-related green fluorescence were measured using the MicroImage Olympus Optical analysis system. The data are presented as Mean±SEM. Bars with different letters denote statistical differences at P<0.05.</p

Analysis of development of cattle embryos co-cultured with BOECs at control (38.5°C) and elevated (41°C) temperatures for 168 h post fertilization.

<p>Data presented as Mean±SEM. Bars with different letters denote statistical differences at P<0.01.</p

Analysis of HSP70 in BOECs cultured alone and co-cultured with cattle embryos at control (38.5°C) and elevated (41°C) temperatures for 168 h post fertilization.

<p>A) Western blots of HSP70 protein. B) Densitometric quantification of HSP70 protein expression. Data presented as Mean±SEM. Bars with different letters denote statistical differences at P<0.05.</p

Fluorescent analysis of OVGP1 in BOECs cultured alone and co-cultured with cattle embryos at control (38.5°C) and elevated (41°C) temperatures for 168 h post fertilization.

<p>A) Representative images of immunofluorescent staining of OVGP1 in BOECs cultured alone at control (38.5°C) (I) and elevated (41°C) (II) temperatures, or co-cultured with cattle embryos at control (38.5°C) (III) and elevated (41°C) (IV) temperatures. Nuclei were counterstained with Hoechst 33342 (blue); OVGP1 localization was determined using antibody against OVGP1 and Alexa Fluor 488-conjugated secondary antibody (green). B) The IOD values of OVGP1-related green fluorescence were measured using the MicroImage Olympus Optical analysis system. The data are presented as Mean±SEM. Bars with different letters denote statistical differences at P<0.05.</p