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Heutelbeck A.,University of Veterinary Medicine Hannover | Oldenhof H.,University of Veterinary Medicine Hannover | Rohn K.,University of Veterinary Medicine Hannover | Martinsson G.,National Stud Lower Saxony | And 2 more authors.
Reproduction in Domestic Animals | Year: 2015

Contents: Equipment for cryopreservation of stallion sperm is not always available. In such cases, diluted semen can be shipped to a facility for later cryopreservation. The aim of this study was to evaluate if selection of sperm via density centrifugation yields higher survival rates when cryopreservation is to be delayed (i.e. carried out after 1 day of storage at 5°C). Two-layer iodixanol as well as single-layer Androcoll density centrifugation were tested and compared with samples prepared with standard centrifugation. Special emphasis was placed on comparing centrifugation on the day of semen collection with centrifugation after 1-day refrigerated storage. Sperm morphology and motility as well as membrane and chromatin integrity were evaluated before and after centrifugation. Sperm motility and membrane integrity were also assessed after cryopreservation. It was found that both two- and single-layer density centrifugation processing resulted in higher percentages of morphologically normal and motile sperm with higher membrane and chromatin integrity, as compared to standard centrifugation or diluted samples. Differences were only in the order of magnitude of 5%. Recovery rates after density centrifugation were only approximately 30-40%. When cryopreservation was carried out after 1-day refrigerated storage, centrifugation processing of sperm directly after semen collection resulted in higher percentages of plasma membrane intact sperm post-thaw as compared to performing centrifugation processing of stored sperm just prior to cryopreservation. No significant differences in progressively motile sperm post-thaw were seen. Taken together, for delayed cryopreservation, it is best to perform density centrifugation directly after collection rather than immediately prior to cryopreservation. © 2014 Blackwell Verlag GmbH. Source

Oldenhof H.,University of Veterinary Medicine Hannover | Heutelbeck A.,University of Veterinary Medicine Hannover | Blasse A.-K.,University of Veterinary Medicine Hannover | Bollwein H.,University of Veterinary Medicine Hannover | And 4 more authors.
Reproduction, Fertility and Development | Year: 2015

The aim of this study was to evaluate inter-individual variability in osmotic properties of stallion spermatozoa and its correlation with cryosurvival. In addition, temperature dependency of hypo-osmotic tolerance and membrane fluidity were studied. Stallion sperm membranes exhibited good resistance towards hypotonic stress in the 15-30°C temperature range, whereas membrane stability was found to be decreased at 4 and 37°C. Bull spermatozoa showed greater hypo-osmotic tolerance compared with stallion spermatozoa, especially at temperatures above 30°C, which coincided with decreased membrane fluidity of bovine spermatozoa in this temperature range. The critical osmolality at 22°C, at which half of the sperm population survived exposure to hypotonic saline solution, was found to vary between 55 and 170mOsmkg-1 among different stallions. Clear correlations were found for pre-versus post-freeze sperm motility and membrane integrity. Pre-freeze percentages of membrane-intact spermatozoa after exposure to hypotonic stress showed a weak correlation with sperm motility after cryopreservation. This correlation, however, was not found when data were corrected for initial numbers of membrane-intact spermatozoa in the sample. We thus conclude that studies on pre-freeze tolerance towards hypotonic stress cannot be used to predict sperm cryosurvival rates for individual stallions. © 2015 CSIRO. Source

Hoffmann N.,University of Veterinary Medicine Hannover | Oldenhof H.,University of Veterinary Medicine Hannover | Morandini C.,National Stud Lower Saxony | Rohn K.,University of Veterinary Medicine Hannover | Sieme H.,University of Veterinary Medicine Hannover
Animal Reproduction Science | Year: 2011

Cryopreserved stallion sperm displays a high degree of male-to-male variability with respect to cell viability after thawing. Animals that have semen with low viability after cryopreservation are classified as 'poor' freezers, and when post-thaw viability is high they are designated as 'good' freezers. Cryoprotective agents that are used for cryopreserving stallion sperm include glycerol, ethylene glycol, methyl formamide, and dimethylformamide, and are typically used in concentrations ranging from 1% to 4%. The aim of this study was to evaluate the osmotic stresses that stallion sperm is exposed to during cryopreservation, and to determine if sperm from 'good' and 'poor' freezers show differences in osmotic tolerance limits and in the suitability of cryoprotective agents. Concentrations of 2-3% of the above mentioned cryoprotectants with freezing extender osmolalities ranging from 580 to 895mOsmkg-1 showed the highest motility rates after freeze-thaw, both for 'good' and 'poor' freezers, for all cryoprotectants tested with slightly higher values for glycerol. Freeze-thawed semen from 'poor' freezers was found to have a lower percentage of progressively motile sperm compared to that of 'good' freezers. Assessment of plasma and acrosomal membrane integrity after return to isosmotic conditions revealed that cryopreserved sperm from 'poor' freezers showed lower osmotic tolerance limits as compared to sperm from 'good' freezers. Semen from 'poor' freezers that was frozen using freezing extenders supplemented with more then 2% cryoprotectant showed decreased viability and increased acrosome reaction upon return to isoosmotic conditions, whereas 'good' freezers could withstand cryoprotectant concentrations up to 3% before a decline in viability was observed. © 2011 Elsevier B.V. Source

Schonbom H.,University of Veterinary Medicine Hannover | Kassens A.,University of Veterinary Medicine Hannover | Hopster-Iversen C.,University of Veterinary Medicine Hannover | Klewitz J.,University of Veterinary Medicine Hannover | And 5 more authors.
Theriogenology | Year: 2015

Pregnancy diagnostics in equine reproduction are routinely performed using transrectal ultrasonography, although it is also possible to visualize the fetus by transabdominal ultrasound examinations from the 90th day of gestation onward. We hypothesized that ultrasound examinations may stress the mare and that the gestational stage status and lactation may influence the mare's stress reaction. To investigate the stress reaction, 25 thoroughbred mares of different age, pregnancy and lactational status underwent a transrectal examination. In pregnant mares, an additional transabdominal examination was performed. Salivary cortisol concentration, mean heart rate, and heart rate variability of mares were assessed to evaluate the reactions of hypothalamic-pituitary-adrenal (HPA) axis and of the autonomic nervous system. Significant differences were observed between lactating and nonlactating mares; with a lower responsiveness to stress in lactating mares. The transrectal ultrasound examination in nonlactating mares induced a significant increase in salivary cortisol (P<0.05), and in the heart rate variability parameter, ratio of low to high frequencies (P<0.05). This reflects an activation of the HPA axis and a shift to more sympathetic dominance. In contrast, a transabdominally performed pregnancy check did not induce an activation of the HPA axis over basal level but increased the mean heart rate and low to high frequency ratio. The results of this study indicate that checks of advanced pregnancies can be easily performed by transabdominal ultrasonography. With regard to animal welfare, this technique should be preferred during midgestation in nonlactating mares. © 2015 Elsevier Inc. Source

Giesecke K.,University of Veterinary Medicine Hannover | Hamann H.,University of Veterinary Medicine Hannover | Stock K.F.,University of Veterinary Medicine Hannover | Klewitz J.,University of Veterinary Medicine Hannover | And 3 more authors.
Animal Reproduction Science | Year: 2011

The research of fertility in humans and other mammals has strongly advanced in the recent years. The examination of molecular mechanisms influencing horse fertility is relatively recent. We chose the angiotensin converting enzyme (ACE), the sperm autoantigenic protein 17 (SP17) and the follicle stimulating hormone (FSHB) as candidates for determining stallion fertility and to analyze associations of intragenic single nucleotide polymorphisms (SNPs), flanking microsatellites and candidate-gene linked haplotypes with the pregnancy rate per oestrus (PRO) in 179 Hanoverian stallions. Fertility traits analyzed were the least square means of PRO for stallions (LSMs) and the paternal and embryonic component of breeding values for PRO (BVs). We detected nine SNPs and two flanking microsatellites in ACE, eight SNPs and two flanking microsatellites in SP17 and four SNPs and one flanking microsatellite in FSHB. Three SP17-associated SNPs and the two flanking microsatellites showed significant association with the embryonic component of BVs and one SP17-associated microsatellite was also significantly associated with the paternal component of BVs. Two ACE-associated SNPs were significantly associated with the embryonic component of BVs. Significantly associated haplotypes were shown for all three candidate genes and the tested fertility parameters. The final regression analysis model indicated that haplotypes of all three candidate genes significantly contributed to the paternal and embryonic fertility components of PRO. This is the first report of associations of ACE, SP17 and FSHB with fertility traits of stallions. © 2011 Elsevier B.V. Source

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