Search

Semen extenders usually contain antibiotics with the aim to minimize bacterial growth, but the indiscriminate use of antibiotics increases the emergence of multidrug-resistant bacteria. A limiting factor of semen processing in dogs is the low total sperm count that limits the number of insemination doses that can be obtained from one ejaculate. Therefore, two ejaculates collected at a short interval can be combined to increase the number of AI doses. In this study, semen was collected from dogs either once or the same dogs (n = 28) were submitted to dual semen collection 1 h apart. All ejaculates were submitted to bacteriological analysis. We hypothesized that bacterial contamination of semen is low but that a dual semen collection might increase contamination. A sample for bacteriological examination was taken from raw semen immediately after semen collection. Bacteria including mycoplasmas were isolated using conventional cultivation procedures and isolates were identified to the species level by matrix-assisted laser desorption ionization - time of flight (MALDI-ToF) mass spectrometry. In total, 22 bacterial species were identified in the 84 ejaculates with Mycoplasma cynos, Streptococcus canis and Canicola haemoglobinophilus being most frequent. Bacterial growth was sporadic in 16 and absent in 10 ejaculates. The overall bacterial growth was lower in the second than in the first ejaculate of dual semen collections (p < 0.05). The percentage of motile and membrane-intact spermatozoa in frozen-thawed ejaculates was not associated with the degree of bacterial contamination of raw semen. In conclusion, there was only limited microbial contamination in dog semen and the microorganisms isolated are considered part of the normal genital bacterial flora. Repeated semen collection reduced bacterial contamination in the second in comparison to the first ejaculate. The use of antibiotics in canine semen should be questioned., Competing Interests: Declaration of competing interest None of the authors has any conflict of interest to declare., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aimed to investigate the effects of storing horse semen either in a dry shipper (≤ -150 °C) or on dry ice (≤ -78 °C) for up to 14 days. A total of 264 frozen semen straws from male horses (n = 8) stored in liquid nitrogen were transferred on day 0 (d0) to a dry shipper or a dry ice styrofoam box. On d1, d3, d7, d10, and d14, straws from the dry shipper and dry ice were returned to the liquid nitrogen container. Semen was evaluated by CASA for total (TMot), progressive motility (PMot) and sperm velocity parameters, by fluorescence microscopy for percentage of membrane-intact sperm (SYBR14/PI), high mitochondrial membrane potential (HMMP; JC1) and DNA fragmentation. Temperature inside the containers was monitored continuously. Until d7, no changes were observed in TMot, PMot, and membrane-intact spermatozoa. Thereafter, all three parameters decreased in semen stored on dry ice but not in a dry shipper (time p < 0.001, time x shipping device p < 0.001). The HMMP decreased continuously over time in both containers with a more pronounced decrease on dry ice compared to the dry shipper (shipping device p < 0.01, time p < 0.001, time x device p < 0.001). The DNA fragmentation increased on d10-14 on dry ice and d14 in the dry shipper (time p < 0.001, time x device p < 0.01). In conclusion, frozen horse semen can be safely stored for up to 7 days on dry ice. Sperm DNA integrity and HMMP, however, were adversely affected after 14 days in both shipping devices., Competing Interests: Declaration of Competing Interest The authors state that there is no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In stallions temporarily not intended for breeding, reversible suppression of testicular function by vaccination against GnRH can be of interest. In the present study, effects of GnRH agonist treatment on the resumption of testicular function after GnRH vaccination were investigated. Testis size, testosterone release, semen characteristics and behavior were evaluated. We hypothesized that GnRH agonist treatment would restore testicular function. Shetland stallions were assigned to an experimental and a control group (n = 6 each). Experimental stallions were GnRH-immunized twice, four weeks apart. Ejaculates for semen analysis and blood for analysis of testosterone concentration and GnRH antibody titers were collected. Each experimental stallion was hemicastrated together with an age-matched control animal when testosterone concentration decreased below 0.3 ng/mL. Three weeks thereafter, daily treatment with the GnRH agonist buserelin was initiated (4 μg/day for 4 weeks followed by 8 μg/day). The remaining testicle was removed when testosterone concentration exceeded 0.5 ng/mL in vaccinated stallions. Time from exposure to a mare until mounting increased in GnRH-vaccinated stallions and decreased with buserelin treatment. Total sperm count decreased after vaccination but increased only slightly in response to buserelin. Sperm motility and percentage of membrane-intact spermatozoa decreased after vaccination and returned to pre-vaccination values with buserelin treatment. Testosterone concentration and testis volume decreased after GnRH vaccination and started to increase with buserelin treatment. In conclusion, the downregulation of testicular function by GnRH vaccination can be counteracted with buserelin. This approach may be useful in GnRH-vaccinated stallions with prolonged suppression of testicular function., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In horse breeding, quality assessment of semen before insemination is often requested. Non-laboratory-based techniques for objective analysis of sperm motility are thus of interest. The aim of this study was evaluating a portable device for semen analysis (Ongo sperm test) and its comparison with computer-assisted semen analysis (CASA). Semen was collected from 10 stallions, diluted to 100, 50 and 25 × 10 6  sperm/ml and analysed for total (TM) and progressive motility (PM). The final sperm concentration influenced total motility analysed by Ongo (p < 0.05) which was higher at 100 × 10 6  sperm/ml when compared to 25 × 10 6  sperm/ml (p < 0.05) but not when compared to 50 × 10 6  sperm/ml (n.s.). Sperm concentration did not influence total motility when assessed by SpermVision (n.s.). Agreement between methods was evaluated by correlation analysis and Bland-Altman plot. Intra-assay variation of Ongo was 5.2% ± 3.0 for TM and 6.9% ± 3.4 for PM. Correlation between Ongo and CASA was r = 0.79, 0.88 and 0.83 for 100, 50 and 25 × 10 6  sperm/ml for TM, and r = 0.87, 0.89 and 0.87 for PM, respectively (all p < 0.001). At the 100 and 25 mio/ml dilutions, the difference between the two systems deviated significantly from 0, while no such bias existed at the 50 mio/ml dilution (TM Ongo 85.0%, CASA 82.3%; PM Ongo 64.1%, CASA 66.1%). The 95% confidence interval was 19.9%, 18.9% and 19.2% ± mean for TM and 20.7%, 17.4% and 20.3% ± mean for 100, 50 and 25 × 10 6  sperm/ml, respectively. In conclusion, Ongo sperm test sperm motility data were strongly correlated with data obtained by CASA. In addition, at a concentration of 50 × 10 6  sperm/ml values measured with both systems were close to identical. At this concentration, which is recommended in equine AI, Ongo and CASA can be used interchangeably., (© 2018 The Authors. Reproduction in Domestic Animals Published by Blackwell Verlag GmbH.)

Increasing day length in spring stimulates reproductive functions in horses. In this study, we have analysed the effect of artificial long days on the quality of cooled-stored and cryopreserved semen in Shetland stallions. Stallions of the treatment group (AL, n = 8) were exposed to 16 h light and 8h darkness from 15th December to 20th March while control stallions (CON, n = 7) were kept under natural photoperiod. Semen was collected once weekly and processed for cooled-storage and cryopreservation once per month. Total and progressive motility and percentage of membrane intact spermatozoa were analysed at 24, 48 and 72 h of cooled-storage and after freezing-thawing, respectively. Total and progressive motility and membrane integrity decreased during cooled-storage for 72 h in each month and both groups (p < 0.001). All these parameters were lower in CON versus AL stallions (p < 0.05) and the decrease was more pronounced in group CON (storage time x group p < 0.05). Differences between groups decreased throughout the observation period from January (p < 0.05 between groups) to July (e.g. total motility after 72 h of cooled-storage in January for group AL 80 ± 3 and group CON 49 ± 12%, respective values in July, 83 ± 2 and 72 ± 6%). Neither total and progressive motility nor percentage of membrane-intact and morphologically defect spermatozoa in frozen-thawed semen differed between groups and months. In conclusion, motility of cooled-stored semen was reduced in January and increased in stallions kept under a long day light programme for at least 30 days., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Horses are seasonal breeders, and robust breeds may exhibit a winter hypometabolism when kept under semiferal conditions. In this study, we analyzed the effects of artificial long days on rectal temperature, heart rate, heart rate variability, hematology, coat changes, semen parameters, and plasma testosterone concentrations in Shetland stallions stabled overnight and assigned to a control group (CON, n = 9) kept under natural photoperiod, and a treatment group exposed to a long-day light program from 15 December to 20 March (AL, n = 9). During the 8-month study, rectal temperature, heart rate, and heart rate variability at no time differed between groups. Plasma total protein (P < 0.05), hematocrit, leukocyte, and lymphocyte counts (P < 0.001) first increased and then decreased during the study period but did not differ between groups. Length of the guard hair decreased over time (P < 0.001) and this decrease occurred earlier in AL than in CON stallions (time × group P < 0.001). Hair regrowth was faster in CON than in AL stallions (over time P < 0.001, time × group P < 0.001). Total sperm count increased from January to April (AL) and May (CON; P < 0.001) but did not differ between groups. Sperm motility and percentage of membrane-intact spermatozoa showed no clear seasonal changes and semen parameters did not differ between groups. In conclusion, Shetland stallions showed seasonal variations in hair coat and total sperm count but only changes in hair coat but not semen parameters were advanced by a long-day light program., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study was undertaken to investigate the effects of storage of stallion semen in a defined milk protein extender at 5 and 15 degrees C under either anaerobic or aerobic conditions, with or without addition of the antibiotic gentamicin. Semen samples were collected from eight fertile stallions and stored for 96 h (day 0-4) and assessed daily for motility, velocity and membrane integrity (viability) using a CASA system. Samples for bacteriology assessment were taken on day 2 of storage. No significant (p > 0.05) differences in motility, velocity or viability were observed between treatments on days 0-2. On days 3 and 4, semen stored without gentamicin at 5 degrees C had a significantly (p < 0.05) better semen quality compared with storage at 15 degrees C without gentamicin, irrespective of oxygen exposure. On days 3 and 4, motility and velocity were greater in samples stored at 15 degrees C with gentamicin, compared with the corresponding treatments without antibiotic (p < 0.05). This effect was also evident for viability on day 4. The decline in semen quality observed at 15 degrees C most likely resulted from the effect of bacterial growth. Bacterial growth was the greatest in samples stored at 15 degrees C without gentamicin, under both anaerobic and aerobic conditions (p < 0.05). Bacterial growth was inhibited by adding of gentamicin at 15 degrees C, which accordingly reduced the decline in semen quality. Addition of antibiotic to samples stored at 5 degrees C had no significant effect on any parameter analysed. In conclusion, storage at 15 degrees C can be achieved by using an extender containing the antibiotic gentamicin. Storage at 5 degrees C tended to maintain better semen quality irrespective of oxygen exposure, and did not necessitate an antibiotic treatment.

Sperm cell membranes are susceptible to peroxidative damage by an excess of reactive oxygen species (ROS). Antioxidative defence systems consisting of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) physiologically control the balance between ROS production and neutralization. In the present study the hypothesis was tested that lipid peroxidation occurs during storage of semen at 5 degrees C and that semen extender has positive effects on the antioxidative potential of equine semen. The aim of the study was to determine the activity of GSH-Px, SOD and CAT and the concentration of thiobarbituric acid reactive substances (TBARS) as an indicator of lipid peroxidation in native semen and after addition of extender, cooling and storage. Semen was collected from fertile Shetland stallions. In experiment 1, activity of antioxidative enzymes was determined immediately after semen collection and after 24 h storage at 5 degrees C. Enzyme activities were measured in native semen, semen diluted with semen extender, spermatozoa resuspended after centrifugation in extender and 0.9% NaCl as well as in undiluted and extender-diluted seminal plasma. In experiment 2, TBARS concentrations were analysed during storage of semen at 5 degrees C for 24 h. Semen storage for 24 h at 5 degrees C did not change activity of the examined enzymes. Antioxidative activity was significantly higher in extended than in native semen as well as in extended plasma than in undiluted plasma. In conclusion, the addition of semen extender increases the antioxidative activity in seminal plasma of stallions. Basal antioxidative activity in native semen as well as increased activity in extended semen are maintained over 24 h storage at 5 degrees C. TBARS content did not increase during semen storage. In conclusion, lipid peroxidation does not increase substantially during semen storage. The enzymatic antioxidative activity in semen apparently prevents ROS formation and is further increased by addition of semen extender.

Numerous diseases are carried and can be transmitted from the African buffalo (Syncerus caffer) to livestock. Buffaloes free of specific diseases (BFSD) are thus in demand amongst game farmers. Current BFSD derive from a small genetic pool and hence there is a special interest in bringing new genetic material into such herds. In this study epididymal sperm from 16 mature African buffalo bulls was frozen with Triladyl and AndroMed extender (Minitüb, Tiefenbach, Germany) with and without addition of bovine seminal plasma. Post-thaw motility, longevity and acrosomal integrity were compared. In all but one animal, post-thaw motility was higher, although not always significant, if sperm was frozen with Triladyl than with AndroMed. Seminal plasma was detrimental to the post-thaw motility. Neither semen extender nor seminal plasma had an influence on post-thaw acrosomal integrity. It can be concluded that bovine seminal plasma at a concentration of 10% is detrimental rather than beneficial for the post-thaw motility of African buffalo sperm. Even though being inferior AndroMed does, however, have the advantage that it is a defined semen extender and therefore clearly has a lower risk of contamination.