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Abstract During semen processing for cryopreservation, most seminal plasma is usually removed, and components with protective effects on sperm may be missing after thawing and within the female reproductive tract. The present study evaluated the effect of postthaw addition of autologous seminal plasma on motion characteristics of stallion sperm with fair (n = 4) or poor (n = 3) freezability. Therefore, pure seminal plasma (group SP1), seminal plasma combined with fresh semen extender (group SP2), or seminal plasma mixed with freezing extender (group SP3) were used to fill 0.5 mL straws and frozen similar to stallion semen. Postthawing, semen samples (n = 42) were diluted either with semen extender (group FT) or with seminal plasma (n = 126) of groups SP1 to SP3 to 25 × 106 sperm/mL. In fair freezer stallions, total and progressive motilities were higher in group FT than in group SP1 (P <.05), but there was no difference in poor freezing stallions among groups (P >.05). However, comparing individual stallions, positive effects of seminal plasma on total or progressive motility were detected in two stallions. Curvilinear velocity increased in groups SP2 and SP3 in fair freezer stallions and in all groups with seminal plasma compared with group FT in poor freezer stallions (P <.05). Although straightness was higher in groups SP2 and SP3 compared with group FT in fair freezer stallions (P <.05), there was no difference among groups in stallions with poor freezability (P >.05). Average lateral head displacement did not change among groups of fair freezer stallions (P >.05) but was higher in groups SP2 and SP3 than in group FT in poor freezer stallions (P <.05). Beat cross frequency was higher in all groups diluted with seminal plasma postthawing in fair freezer stallions (P <.05), but only in group SP1 than in group FT in poor freezer stallions (P <.05). The addition of autologous seminal plasma to frozen-thawed semen can improve motion characteristics of stallions with fair and poor freezability. This is a valuable additional protocol for laboratories dealing with cryopreservation of stallion semen and for veterinarians working with fair or poor freezer stallions. Highlights • Autologous seminal plasma improved sperm motion in fair and poor freezer stallions. • Twenty-five percentage of seminal plasma added to frozen-thawed semen was beneficial compared with 50%. • Fresh semen extender with seminal plasma was favorable to frozen semen extender. • Postthaw sperm parameters were not comparable to good freezer stallions. [ABSTRACT FROM AUTHOR]

Abstract The aim of the present study was the investigation of stallion epididymal sperm frozen with seminal plasma within the same straw separated by an air bubble before freezing. We hypothesized that mixing both components after thawing improves the sperm quality compared with conventionally frozen-thawed epididymal sperm. Cryopreservation of epididymal sperm allows the preservation of spermatozoa in case of death or termination of the breeding carrier. However, these sperm lack contact with the seminal plasma, which plays an important role in the development of sperm functionality and the regulation of uterine inflammatory response after insemination. Motion characteristics, morphology, and viability were assessed in epididymal sperm mixed with seminal plasma after thawing (group SP) and compared with conventionally packed cryopreserved epididymal sperm that had no contact with seminal plasma (group FT). Total and progressive motility, curvilinear velocity (VCL), and average lateral head displacement were significantly higher in group SP (P <.05). During post-thaw incubation, VCL decreased in group FT but not in group SP (P <.05), and beat cross-frequency was significantly lower in group FT than in group SP after post-thaw incubation (P <.05). Straightness did not differ between the groups (P >.05). Although the percentage of tail abnormalities and isolated sperm heads increased significantly only in group SP after cryopreservation (P <.05), the proportion of morphologically abnormal and nonviable sperm was not affected by seminal plasma (P >.05). Mixing both components after thawing increased sperm motility and velocity but did not negatively affect morphology or viability. This packaging system is promising for routine artificial insemination of stallion epididymal sperm. Highlights • Post-thaw addition of seminal plasma improved motility in epididymal sperm. • Post-thaw addition of seminal plasma improved VCL and ALH in epididymal sperm. • Separated seminal plasma avoids its negative effects during cryopreservation. • Combined packaging of sperm and seminal plasma is promising in equine breeding. [ABSTRACT FROM AUTHOR]

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To preserve epididymal sperm, only a limited number of sperm is available, and an optimum processing method that is applicable for the majority of stallions is therefore crucial for successful preservation. The aim of the present study was to evaluate the effect of four different extenders that are commercially available for chilled semen on the motion characteristics of stallion epididymal sperm. Sperm were harvested by retrograde flush from 20 epididymides after the routine castration of 10 stallions. Aliquots of sperm samples were diluted with each of the four extenders ([E1] skim milk-based, [E2] containing defined milk protein, [E3] containing egg yolk, and [E4] containing caseinate). Total motility (TMOT %) and progressive motility (PMOT %) assessed immediately after sperm harvesting and during prolonged storage were highest in extenders with selected milk proteins (E2: 54, 24–79 and 50, 19–78; E4: 57, 23–82 and 52, 14–80) compared to the skim milk-based extender (E1: 40, 1–66 and 37, 0–62) and the egg yolk-containing extender (E3: 21, 6–48 and 13, 2–40; median, min–max for TMOT and PMOT, stored for 48 hours at 4°C, respectively). Motility values were similar for extenders E2 and E4 during the entire storage period ( P > .05), while extenders E1 and E3 yielded significantly lower values ( P < .05). Using E1, motility increased during the storage period but did not reach values similar to those of E2 or E4. Curvilinear velocity (VCL), amplitude lateral head displacement (ALH), and beat-cross frequency (BCF) differed among all extenders after sperm harvesting ( P < .05), but not after 24 (VCL, ALH) and 48 hours (VCL, ALH, BCF; P > .05). Based on the motility results, we recommend extenders containing defined milk protein to process epididymal sperm. [ABSTRACT FROM AUTHOR]

Reducing seminal plasma (SP) is essential in stallion semen preservation; however, SP has an important role in sperm fertility as well as sperm protection and transportation in the female genital tract. In the present study, semen storage at high concentrations and low volumes and the effect of SP added immediately before insemination, volume, and deposition (corpus or uterine horn) of the insemination dose were evaluated. Semen processing protocols were investigated in experiment 1: dilution to a final volume of 20 mL (control); or centrifugation and dilution of the sperm pellet to a volume of 1.5 mL containing 500 × 10 6 sperm. Immediately before insemination, centrifuged samples were diluted with INRA96 extender containing 0%, 5%, 20%, or 80% SP to a final volume of 20 mL. In experiment 2, concentrated semen was not further processed before insemination. Pregnancy rates were lowest when adding 20% SP to the semen extender (100%, 89%, 67%, 30%, and 67% for control; 0%, 5%, 20%, and 80% SP, respectively; P < .05). Embryonic growth between days 12 and 14 was highest using 0% SP (292%, 515%, 290%, 343%, and 404% for control; 0%, 5%, 20%, and 80% SP, respectively; P < .05). Uterine fluid accumulation did not differ among groups ( P > .05). The number of progressively motile sperm per dose was similar in pregnant (median, range: 347, 217–452) and nonpregnant cycles (314, 195–369; P > .05). Deep intrauterine insemination did not improve pregnancy outcome when using 500 × 10 6 sperm ( P > .05). Stallion semen can be stored at 5°C for 24 hours highly concentrated with low volumes of extender, yielding normal pregnancy rates. Adding SP to ejaculated sperm immediately before insemination is not necessary after 24 hours of cooled storage. [ABSTRACT FROM AUTHOR]

Insemination with chilled transported semen has become distinctly important in the horse-breeding industry. To ensure cell survival during cooled storage, semen is diluted with an appropriate extender and the concentration of seminal plasma (SP) is reduced. Nevertheless, SP plays an important immunomodulatory role in the female genital tract and supports sperm fertility. The aim of the present study was to evaluate the effect of the addition of autologous SP after cooled storage to highly concentrated stallion semen. Therefore, SP was removed by simple centrifugation of extended semen, aspiration of the supernatant, and resuspension of the sperm pellet with semen extender. Motion characteristics were evaluated after cooled storage for 48 hours at concentrations of 333 × 10 6 sperm/mL in comparison with stored samples at concentration of 25 × 10 6 sperm/mL (control). The highly concentrated semen samples were diluted with an extender containing 0%, 5%, 20%, and 80% SP directly before motility analysis. Dilution of the cooled semen with a fresh semen extender without SP (0%) increased kinematic parameters (curvilinear velocity [VCL] 137.3 vs. 151.8; straight-line velocity [VSL] 49.0 vs. 57.5; average path velocity [VAP] 69.5 vs. 79.4 μm/second; amplitude of lateral head [ALH] 3.1 vs. 3.3 μm; beat cross frequency [BCF] 31.6 vs. 33.5 Hz; P < .05) but not total motility (51% vs. 43%) and progressive motility (46% vs. 36%) compared with controls. The addition of SP after storage for 48 hours decreased sperm total motility and progressive motility regardless of SP concentration: 5 (38% and 34%), 20 (37% and 33%), and 80% SP (27% and 22%; P < .05). In contrast, kinematic parameters were enhanced by extenders containing 5% and 20% SP (VCL: 148.0 and 155.6; VSL: 59.2 and 60.9; VAP: 78.7 and 81.9; BCF: 33.4 and 35.7; ALH: 3.4 and 3.4; P < .05). However, using an extender containing 80% SP was detrimental to kinematic parameters (VCL: 151.2; VSL: 52.2; VAP: 76.9; BCF: 34.8; P < .05) except for ALH, which increased (3.5; P < .05). In conclusion, cooled storage at concentrations of 333 × 10 6 sperm/mL did not affect sperm motility. The addition of a fresh extender or an extender containing small concentrations of SP to highly concentrated ejaculated sperm increased kinematic values after storage; however, increasing concentrations of SP decreased sperm motility. [ABSTRACT FROM AUTHOR]

Semen cryopreservation is of growing interest in the horse breeding industry, and collecting epididymal sperm might provide the chance to preserve genetic material from valuable stallions after severe injury or death. In case of an unexpected emergency, there may not always be an adequate laboratory nearby. Therefore, we compared fast and slow freezing methods using either a programmable freezer or a styrofoam box filled with liquid nitrogen. Epididymides of 10 stallions were collected immediately after routine castration under general anesthesia. Epididymal spermatozoa were evaluated before and after the freeze-thaw process for motility, viability, morphological, and kinematic parameters. Neither postthaw motility nor kinematic values differed among the four freezing protocols. Morphological abnormalities after freezing and thawing differed among epididymal segments. However, there were significantly more nonviable spermatozoa after the freeze-thaw process using the fast freezing process in the styrofoam box filled with liquid nitrogen compared with all other freezing processes. According to the results of this study, freezing in nitrogen vapor should be considered as an alternative to the programmable freezer only in combination with a prolonged cooling period. [ABSTRACT FROM AUTHOR]

Abstract: Preservation of epididymal spermatozoa is an advantageous method to preserve genetic material of endangered species or valuable breeding animals after sudden death and injuries. Despite lower pregnancy rates, fertilization with epididymal sperm has been proven successful. Variable sperm quality after cryopreservation among individual stallions and the usually terminal chance to preserve epididymal sperm are opportunities for the development of a freezing procedure suitable for the majority of stallions. To evaluate the effect of the preservation procedure, we analyzed the sperm motion characteristics after every step of processing. In addition, we investigated the influence of seminal plasma on motility of frozen-thawed semen. We compared three segments of the cauda epididymidis and harvested spermatozoa by retrograde flushing (most caudal part) and mincing (more cranial segments) to augment the number of spermatozoa. During processing, there were differences in sperm motion characteristics depending on the segment of the cauda epididymidis. Distinct increases in motility due to different extenders and the effect of seminal plasma suggest that motion characteristics of raw and frozen-thawed spermatozoa are strongly influenced by microenvironment and must be interpreted with caution. [Copyright &y& Elsevier]

The aim of the present study was to evaluate the effect of different freezing procedures on sperm motion, viability, the acrosome status, mitochondrial membrane potential (MMP), intracellular calcium content, and DNA integrity on epididymal stallion sperm. Therefore, the sperm of 10 healthy stallions was harvested by retrograde flushing after testectomy, diluted with a semen extender containing defined milk proteins and a freezing extender containing egg yolk and glycerol and frozen according to 4 different protocols, using a programmable freezer and a floating rack performing a slow (processes 1 and 2) or a fast cooling rate (processes 3 and 4, respectively). Post-thaw total motility and slow sperm values were lower when using process 4 compared with processes 1 and 2 (P <.05) after 1 hour of incubation. Progressive motility was lower in process 4 compared with process 1 immediately after thawing and after 1 hour of incubation (P <.05). The amount of rapid sperm was lower when using process 4 compared with process 1 immediately after thawing (P <.05). After 1 hour of incubation, the amount of rapid sperm was lower when using process 4 compared with processes 1 and 2 (P <.05). Higher values for viable sperm were seen in processes 1 and 2 compared with process 4 (P <.05) after 1 hour of incubation. Immediately after thawing, more viable sperm with high MMP (hMMP) were observed when using process 3 compared with process 2 (P <.05). After 1 hour of incubation, a significantly higher amount of viable hMMP sperm were detected when using processes 1 and 2 compared with process 4 (P <.05). Process 2 yielded a lower percentage of sperm containing low calcium (lCa) than process 3 immediately after thawing (P <.05). After 1 hour of incubation, the lowest amount of lCa sperm was observed using process 4 (P <.05). The subpopulation of viable/hMMP/lCa sperm was higher when using process 3 compared with process 2 immediately after thawing (P <.05). After 1 hour of incubation, the lowest amount of this subpopulation was detected in process 4 (P <.05). The DNA integrity was similar in all groups. In conclusion, a slow cooling rate with a controlled rate freezer resulted in best sperm quality after thawing. Using a floating rack in nitrogen vapor as an alternative to a programmable freezer, equilibration in a cooled environment is advantageous. • Freezing on the floating rack without precooling resulted in lowest sperm quality. • Cooling the sperm before freezing on the floating rack improved sperm quality. • Slow cooling with a controlled rate freezer resulted in the best sperm quality. [ABSTRACT FROM AUTHOR]

Abstract: Treatment with the progestin altrenogest is widely used in pregnant mares. The fact that foals born from healthy mares treated with altrenogest until term suffered from neonatal problems raises the question of direct effects of altrenogest on vital functions in the neonate. We have therefore investigated altrenogest concentrations in maternal and neonatal blood plasma and in fetal fluids. Pregnant mares were treated with altrenogest orally once daily (0,088 mg/kg bodyweight, n = 7) or left untreated (n = 8) from 280 d of gestation until foaling. Altrenogest concentration was determined in plasma of the mares, their foals and in amniotic and allantoic fluid. The concentration of altrenogest in plasma from treated mares (2.6 ± 1.0 ng/mL) was significantly lower than in plasma from their foals immediately after birth (5.6 ± 1.9 ng/mL; p < 0.05), but was significantly higher than in their fetal fluids (amniotic fluid: 0.4 ± 0.1 ng/mL; p < 0.05; allantoic fluid: 3.0 ± 1.5 ng/mL). Altrenogest was undetectable in maternal and fetal plasma and fetal fluids of control pregnancies at all times. Altrenogest concentration in plasma of foals from treated mares was strongly correlated to the altrenogest concentration in plasma of their dams (r = 0.938, p < 0.001) and in amniotic (r = 0.886, p < 0.001) and allantoic fluid (r = 0.562, p < 0.05). A significant decrease in altrenogest concentration between the time periods 0–15 min, 30–120 min, and 180–360 min after parturition was seen in the plasma from foals born to altrenogest-treated mares. In conclusion, our data demonstrate that altrenogest reaches the equine fetus at high concentrations. [Copyright &y& Elsevier]