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1.
The dilution effect and effect of restoring seminal plasma (SP) proportion in diluted semen were determined in chilled Asian elephant sperm. Semen was collected from eight males, and samples with ≥30% motile sperm were used in the study. Tris‐glucose‐egg yolk extender (TE) was used for cooled storage at 4°C for 48 hr. In experiment 1 (n = 18), semen was diluted to 1:1, 1:3, 1:7 and 1:15 with TE (volume per volume). There were no significant changes in sperm viability and sperm with normal acrosome integrity among dilutions, but sperm motility and motility velocities were greater (p < .05) in the 1:1 dilution than those of the 1:7 and 1:15 dilutions at 48 hr of storage. In experiment 2, supplemented SP was derived from elephants and stallions. In experiment 2.1, diluted semen (1:7 dilution) was restored with SP to obtain a 1:2 proportion (n = 8). Sperm motility, viability and sperm with normal acrosome integrity were similar among treatments, but motility velocities were greater (p < .05) with stallion SP at 48 hr of storage. In experiment 2.2, diluted semen (1:15 dilution) was restored with SP to obtain a 1:3 proportion (n = 10). Sperm viability and sperm with normal acrosome integrity were similar among treatments at 48 hr of storage. However, sperm motility and motility velocities were greater (p < .05) with stallion SP than those of others. In conclusion, elephant sperm motility was affected by a dilution effect and restoration of SP proportion with stallion SP, but not with elephant SP, could improve motility in chilled highly diluted sperm.  相似文献   

2.
Slow-cooled stallion spermatozoa, with and without seminal plasma removed by centrifugation, were diluted in Kenney's extender (KE) containing nonfat dry skim milk with glucose and antibiotics or in KE supplemented by adding a modified high-potassium Tyrode's medium (KMT). Four ejaculates from each of four stallions were collected and divided factorially across these four treatments. Percentage of motile sperm, path velocity, and linearity immediately after treatment (0 h) and after storage at 4 degrees C for 24, 48, and 72 h were evaluated objectively by use of a HTM-2030 sperm motility analyzer. Stallions were a significant source of variation (P less than .01) throughout. After sperm had cooled, effects of stallion, extender, centrifugation, and their interactions were all found to be significant (P less than .01). The motility at 0, 24, 48, and 72 h for centrifuged KE was 74, 47, 39, and 24%; for uncentrifuged KE was 76, 56, 50, and 37%; for centrifuged KMT was 76, 75, 72, and 64%; and for uncentrifuged KMT was 80, 50, 26, and 13%, respectively. The extender x centrifugation interaction, after 24, 48, and 72 h of storage, accounted for half or more of the variation. Whereas centrifugation of semen extended in KE seemed to be harmful to sperm, motility of sperm extended in KMT after centrifugation was remarkably conserved for 72 h and was superior to all other treatments (P less than .05). This extender is promising for preserving liquid stallion semen when it must be transported before use in artificial insemination.  相似文献   

3.
Processing stallion semen for assisted reproductive procedures, such as intracytoplasmic sperm injection (ICSI), requires special considerations regarding cooling, concentrating, and handling of sperm. The aim of experiment 1 was to determine whether cooled semen could be frozen without removal of seminal plasma and at a low sperm concentration while maintaining motile sperm for ICSI selection procedures. In experiment 2, five media for holding stallion sperm were compared to evaluate sperm motility for an interval of time sufficient for ICSI sperm selection procedures. In experiment 1, semen samples from eight stallions were cooled for 24 hours in two extenders, CST (E-Z Mixin-CST “Cool-Store/Transport” Animal Reproduction Systems) and INRA96 (Institut National de la Recherche Agronomique, IMV International Corporation), before being frozen in four freezing diluents, and were evaluated at 0, 45, and 75 minutes after thawing. The cooling extender did not significantly affect sperm motility, but modified French and glycerol egg yolk diluents provided the best sperm motility for frozen–thawed groups. In experiment 2, semen samples from seven stallions were used to test five media for holding sperm. Samples were analyzed for total and progressive motility at hourly intervals. Mean total and progressive motility were not different (P > .05) among groups from 1 through 4 hours. At 5 hours, groups differed (P = .004), with sperm held in Tyrode’s with albumin, lactate, and pyruvate having higher (P < .05) total and progressive motility than all other samples. In conclusion, motile stallion sperm can be obtained after the sperm are cooled for 24 hours, frozen, and thawed; various media are available to maintain sperm motility during equine ICSI selection procedures.  相似文献   

4.
The purpose of this study was to determine the effects of different concentrations of coenzyme Q10 (CoQ10) and α-tocopherol (T) along with their interaction effects on the quality of preserved stallion semen at 5°C for a period of 48 hours. Semen was collected and diluted with skim milk–based extender that was supplemented with different antioxidants: no antioxidant (negative control [NC]), 0.9% (vol/vol) dimethyl sulfoxide (positive control [PC]), α-tocopherol (5 [T5] or 10 [T10] mM), CoQ10 (1 [C1] or 2 [C2] μM), 1 μM CoQ10 + 5 mM α-tocopherol (C1T5), 1 μM CoQ10 + 10 mM α-tocopherol (C1T10), 2 μM CoQ10 + 5 mM α-tocopherol (C2T5), and 2 μM CoQ10 + 10 mM α-tocopherol (C2T10), then kept at 5°C. The results showed that C1 extender resulted in higher total motility (62.44 ± 3.82) and plasma membrane integrity (65.16 ± 3.63%) compared with NC after 48 hours of storage (P < .05). Different concentrations of α-tocopherol had no significant effects on sperm quality, with the exception of plasma membrane integrity, compared with NC and PC extenders (P > .05). Also, C1T5 extender improved total and progressive motility, plasma membrane integrity and functionality, and decreased lipid peroxidation compared with NC and C2T10 extenders over 48 hours of storage at 5°C (P < .05). The C1T5 extender was similar to C1 and T5 extenders in all semen parameters evaluated during storage time. In conclusion, between previously mentioned extenders, C1T5 could improve stallion sperm quality during 48 hours of storage. In the present study, none of extenders had effect on sperm quality until 24-hour storage.  相似文献   

5.
The aim of the present study was to evaluate the quality of raw and cooled semen in Icelandic stallions. Experiments were performed using seven stallions aged between 3 and 19 years. From each stallion, six ejaculates were collected, and semen quality was determined. Thereafter, the semen was split into eight equal parts and processed with and without centrifugation using the extenders INRA 82-egg yolk, INRA 96, GENT, and Equi-Pro to a final concentration of 30 × 106 sperm/mL. The extended semen was then cooled in an Equitainer, where it was stored for 24 hours, and subsequently refrigerated for another 24 hours at 5°C. Immediately after dilution as well as after 24 and 48 hours storage, sperm motility was analyzed using computer-assisted sperm analyzer, and viability was assessed after dual DNA staining with SYBR-14 in combination with propidium iodide. The results show that the stallion had a significant (P < .05) influence on all variables evaluated in raw semen, and mean (±SEM) values of 43.4 ± 4.3 mL for the volume, 193.0 ± 17.0 × 106 sperm/mL for the concentration, 6.7 ± 0.5 × 109 for total sperm and 73.5 ± 2.1% for total sperm motility, 48.7 ± 2.0% for progressive motility, and 65.3 ± 2.0% for rapid cells were measured. In the cold-stored semen, all variables were significantly (P < .05) influenced by the stallion, extender, and storage time (48 hours). Except for Equi-Pro, all extenders examined were suitable for cooled semen preservation. For storage of more than 24 hours, centrifugation and removal of the seminal plasma were advantageous for all extenders with the exception of Equi-Pro.  相似文献   

6.
The present study was conducted with the hypothesis that addition of cholesterol to the extender would stabilize the sperm membranes by increasing the cholesterol-to-phospholipid (C:P) ratio and would result in an improved post-thaw semen quality and reduce oxidative stress in the jack (Martina franca) semen. Forty-eight ejaculates from six jacks were collected and analyzed for the present study. The freshly collected semen sample of each jack stallion was divided into five equal fractions after addition of the primary extender without cholesterol-loaded cyclodextrin (CLC) (C) and with 1, 1.5, 2, and 3 mg/mL CLC to obtain 120 × 106 sperm/mL spermatozoa concentration. The semen was cryopreserved using customized freezing protocols. Evaluation of seminal parameters, the C:P ratio, and the oxidative status of jack spermatozoa was analyzed at all stages of cryopreservation. The oxidative status in the jack semen was evaluated by measuring malondialdehyde, glutathione and total antioxidant capacity levels. The results indicated that the mean percent values for various seminal quality parameters and the oxidative parameters were found to be significantly higher (P < .05) in CLC-treated groups with the highest values for 2 mg of CLC/120 × 106 spermatozoa. In conclusion, the present study revealed that the supplementation of CLC before cryopreservation has significantly reduced the oxidative stress and also increased the C:P ratio during semen cryopreservation process. Furthermore, a reduction in lipid peroxidation levels, reduced damage to the sperm plasma and acrosome membranes and improvement in the post-thaw sperm integrity as well as stability were recorded.  相似文献   

7.
Breeding mares with cryopreserved semen requires specialized equipment for storage and thawing and more intensive mare management. The objectives of this study were (1) evaluate the longevity of frozen stallion semen once it had been thawed, extended, and maintained at 5°C for 48 hours in a passive cooling container, and (2) determine fertility potential of frozen semen that had been thawed, extended, and used to inseminate mares after 24 hours of cooled storage. Eight ejaculates were collected and aliquots were cooled in either INRA96 and CryoMax LE minus cryoprotectant at a concentration of 50 million total sperm/mL. The remainder of the ejaculate was frozen in CryoMax LE extender at a concentration of 200 million total sperm/mL. Semen was thawed using 1 of 3 thawing protocols, and diluted to a concentration of 50 million total sperm/mL in either INRA96 or CryoMax LE minus cryoprotectant and cooled to 5°C. Sperm motility was evaluated at 24 and 48 hours. Eight mares were inseminated over two estrous cycles using frozen semen that had been thawed, extended in INRA96, and cooled for 24 hours. There was no difference in progressive motility at 24 or 48 hours of cooled-storage post-thaw between the 3 thawing protocols. An overall per cycle pregnancy rate of 56% (9/16 cycles) was achieved using frozen-thawed semen that had been extended and cooled for 24 hours. In summary, frozen stallion sperm was thawed, extended, and cooled to 5°C for 24 hours and still maintained adequate (>30%) sperm motility and fertility.  相似文献   

8.
Damage occurring to spermatozoa during cryopreservation results in a loss of motile cells and cells that are functionally normal, compared with fresh sperm samples. Treating bull sperm with cholesterol-loaded cyclodextrins (CLC) before cryopreservation results in increased sperm cryosurvival. However, in previous studies, CLC were always added to sperm samples that had been highly diluted. The aim of this study was to develop a procedure for adding CLC to whole bull ejaculates that would optimize sperm cryosurvival. Adding 2 or 4 mg of CLC/120 x 10(6) sperm to sperm samples ranging in concentration from 120 to 2,000 x 10(6) sperm/mL resulted in greater (17 to 28 percentage points; P < 05) numbers of live cells compared with control samples (no CLC treatment), regardless of the sperm concentration, except for samples at 120 x 10(6) sperm/mL treated with 4 mg of CLC. Incubating sperm with CLC at 23 or 37 degrees C before cryopreservation resulted in similar sperm cryosurvival. The cooling rate used to cryopreserve CLC-treated cells did not affect sperm cryosurvival. Finally, adding CLC to undiluted ejaculates (2 mg of CLC/120 x 10(6) sperm) resulted in greater percentages of live sperm compared with the control samples (62 vs. 45%; P < 0.05), although the percentages of motile sperm were similar for both CLC-treated and control samples (58%). In conclusion, bull sperm cryo-survival can be improved if spermatozoa are treated with CLC before freezing. In addition, CLC can be added to fresh ejaculates at either 23 or 37 degrees C. This technique is simple, practical, and can be easily integrated into current cryopreservation protocols.  相似文献   

9.
Cooling of equine semen obtained from some stallions results in lower seminal quality and viability when the seminal plasma (SP) is present. The objective of this study was to evaluate the effect of the removal of SP using a Sperm Filter on the viability of cooled stallion semen. For this purpose, 31 stallions were used. Their ejaculates were divided into three groups: CN, semen was diluted with an extender; FLT, SP was removed by filtration; and CT, SP was removed by centrifugation and cooled to 15°C for 24 hours. Sperm kinetics and plasma membrane integrity were evaluated immediately after collection (T0) and after 24 hours of refrigeration (T1). No difference (P > .05) was noted at T1 for total sperm motility (TM), progressive sperm motility, or plasma membrane integrity when semen samples from all the stallions were analyzed. However, when samples from stallions termed “bad coolers” were analyzed (TM = <30% at T1), a difference was observed in TM and progressive sperm motility for CN compared with FLT and CT at T1. Sperm recovery was greater when SP was removed using the filter (FLT) to that when the SP was removed by centrifugation (CN) (89% vs. 81%). Thus, we concluded that filtering with a Sperm Filter is an efficient and practical method for removal of SP from stallion ejaculates, with lower sperm loss than centrifugation. We also found that the presence of SP reduces the quality and viability of cooled semen from stallions whose semen is sensitive to the process of refrigeration.  相似文献   

10.
The aim of the present study was to assess the effect of manganese (III) meso-tetrakis (4-benzoic acid) porphyrin (Mn-TBAP) on stallion sperm quality during storage at 5°C. In the present study, 18 ejaculates from three stallions were collected and diluted by INRA82 extender containing 0 (Mn-0), 100 (Mn-100), 200 (Mn-200), and 300 (Mn-300) μM of Mn-TBAP. Sperm motility, plasma membrane integrity and functionality, and lipid peroxidation as indicated by malondialdehyde (MDA) in the spermatozoa of diluted semen were evaluated in vitro at 2, 24, and 48 hours after storage at 5°C. The results showed that all evaluated sperm parameters, except MDA concentration, decreased significantly during the storage period. Total and progressive motility of spermatozoa were higher in Mn-200 extender (46.75 ± 0.58 and 27.62 ± 0.6, respectively) compared with Mn-0 (44.43 ± 0.58 and 25.13 ± 0.6, respectively) and Mn-300 (43.95 ± 0.58 and 25.28 ± 0.6, respectively) after 48 hours of storage at 5°C (P < .05). In addition, sperm plasma integrity and functionality were higher in Mn-200 extender (53.12 ± 0.6 and 46.63 ± 0.78, respectively) compared with Mn-0 (47.74 ± 0.6 and 40.96 ± 0.78, respectively), Mn-100 (48.63 ± 0.6 and 41.99 ± 078, respectively), and Mn-300 (46.11 ± 0.6 and 3.75 ± 0.78, respectively) after 48 hours of storage at 5°C (P < .05). The result showed also that MDA level was lower in Mn-100 extender (3.91 ± 0.06) compared with Mn-0 (4.51 ± 0.06), Mn-200 (4.25 ± 0.06), and Mn-300 (4.75 ± 0.06) after 48 hours of storage at 5°C (P < .05). In conclusion, INRA82 extender supplemented with 200-μM Mn-TBAP could efficiently preserve Caspian stallion spermatozoa after 48 hours of storage at 5°C.  相似文献   

11.
The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 106/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 106 ± 21, 59 × 106 ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.  相似文献   

12.
Thyme (Thymus vulgaris) is a subshrub from the lamiaceae family with plants that are rich in essential oils and antioxidative phenolic substances. The aim of the study was to investigate the effect of dietary thyme and fish oil supplementation on the semen characteristics of miniature Caspian horse. Sixteen stallions were randomly allocated into four groups and received four different diets: unsupplemented control diet, supplemented with fish oil at 2.5% dry matter intake (DMI), supplemented with fish oil (2.5% DMI), and thyme (0.02% DMI), and supplemented with thyme (0.02% DMI). All experimental diets were formulated according to National Research Council (1998). Semen was collected at 0, 30, 60, and 90 days. The semen samples were cooled and preserved at 5°C. Cooled diluted semen samples were evaluated in vitro by microscopic assessments of chilled sperm motility, acrosomal and other abnormalities (head, midpieces, and tail), viability (evaluated by Eosin–nigrosin), and plasma membrane integrity (evaluated by hypo osmolarity swelling test), and the level of malondialdehyde (MDA) was determined during cool storage 0, 24, and 48 hours after collection. The results showed that total and progressive sperm motility and plasma membrane integrity and functionality in all groups were significantly decreased with increasing storage time. On the other hand, the level of MDA in all groups was significantly increased with increasing storage time. Also, the results showed that most sperm quality parameters in this study were significantly higher in fish oil–thyme and fish oil group compared with thyme and control groups after 24 and 48 hours of storage at 5°C. We concluded that dietary supplementation of fish oil and thyme can improve sperm quality in miniature Caspian stallions during storage in cool condition via increasing total and progressive motility and plasma membrane integrity and functionality. More advances in vitro evaluations and artificial insemination are required to reveal the exact effects of thyme on miniature Caspian stallion sperm quality and its fertilizing ability.  相似文献   

13.
The aim of the present study was to evaluate the effect that the addition of cholesterol‐loaded cyclodextrins (CLC) to the thawing extender has on the quality of frozen‐thawed boar sperm. Pooled semen (n = 5) from three boars was used for the experiments. The semen was cryopreserved with an egg‐yolk‐based extender, it was diluted after thawing in Beltsville thawing solution (BTS) supplemented with different concentrations of CLC (0, 12.5, 25, 50 or 100 mg/500 × 106 sperm), and these samples were incubated at 37°C for 150 min. The following parameters of sperm quality were evaluated 30 and 150 min after incubation: sperm with intact plasma membrane (SIPM; %), sperm with normal acrosomal ridge (NAR; %), total motile sperm (TMS; %), progressively motile sperm (PMS; %) and kinetic parameters. Both SIPM and NAR increased (p < 0.05) when the thawing extender was supplemented with 12.5, 25 and 50 mg CLC/500 × 106 sperm. Nevertheless, motility decreased (p < 0.05) when the concentration of CLC exceeded 12.5 mg CLC/500 × 106 sperm. In conclusion, our results suggest that the supplementation of thawing extenders with CLC improves sperm viability and reduces acrosome damage after freezing/thawing.  相似文献   

14.
Captive breeding has become an important tool in species conservations programmes, maintaining genetic diversity and restoring wild, endangered populations. In order to improve the reproductive efficiency of captive kept capercaillie, the purpose of the study was to determine the effect of selenium and vitamin E addition to semen extender on sperm characteristic during short‐term storage. Ejaculates collected individually from four capercaillie were divided into two parts, diluted threefold with basic EK extender and EK enriched with 1 mg/ml of organic selenium and 8 mg/ml of vitamin E (EK+Se+E) and stored 24 hr at temp. +4°C. Spermatozoa morphology, motility and motility parameter were evaluated in net, diluted and stored semen samples. Significant (p < .05) differences between individual males were stated in relation to the majority of traits evaluated in the freshly collected semen. Comparing to the fresh semen, a significant (p < .05) decrease in percentage of live sperm in total (by 3.8% points on average) has been observed in samples diluted by EK extender, while in semen diluted with EK+Se+E extender this decrease was lower (1.5%pts on average) and not significant. Also per cent of motile sperm in EK+Se+E extender was higher (p < .05) then in EK (71.6% vs. 58.9%), but taking into account the values of individual males, both extender and male effect on liquid semen storage become apparent. Obtained data allow concluding that selenium and vitamin E addition to EK extender had positive effect on morphology and motility of capercaillie semen stored 24 hr at 4°C and can be recommended for similar studies carried out on other Galliformes species.  相似文献   

15.
This study was conducted to investigate the effect of glutathione-supplemented INRA82 extender on miniature Caspian stallion sperm quality during storage at 5°C. A total of 12 ejaculates from three stallions (four ejaculates from each stallion) were collected and diluted with INRA82 extender that included different concentrations of glutathione (0 [INRA-G0], 5 [INRA-G5], and 10 mM [INRA-G10]) and stored for 48 hours at 5°C. Sperm motility (computer-assisted sperm analysis), plasma membrane integrity (eosin–nigrosin staining) and functionality (hypo-osmotic swelling test), and malondialdehyde (MDA) level were determined during storage at 5°C. The results showed that the sperm total and progressive motility and plasma membrane integrity and functionality in all extenders were significantly decreased with increasing storage time. However, the MDA level in all extenders was significantly increased with increasing storage time. Also, the results showed that most of the evaluated sperm quality parameters in the present study, with the exception of MDA, were significantly greater in INRA-G5 than in INRA-G0 and INRA-G10 after 24 and 48 hours of storage at 5°C. We have concluded that supplementation of INRA82 with 5 mM glutathione can improve miniature Caspian stallion sperm quality during storage at 5°C by increasing total and progressive motility, plasma membrane integrity and functionality, and decreasing the MDA level compared with INRA-G0 and INRA-G10. More advanced in vitro evaluations and artificial insemination are required to reveal the exact effects of INRA-G5 on miniature Caspian stallion sperm quality and its fertilizing ability.  相似文献   

16.
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 × 106 sperm/mL in comparison with stored samples at concentration of 25 × 106 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 × 106 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.  相似文献   

17.
The objective of this study was to compare semen parameters and embryo recovery rates of cooled stallion semen extended with INRA 96 or BotuSemen Gold. In experiment 1, 45 ejaculates from nine mature stallions were collected, assessed, and equally split between both extenders and then extended to 50 million sperm/mL. Then, the extended semen was stored in three passive cooling containers (Equitainer, Equine Express II, and BotuFlex) for 48 hours. In experiment 2, the same ejaculates extended in experiment 1 were cushion-centrifuged, the supernatant was discarded, and the pellets were resuspended at 100 million sperm/mL with their respective extender. Semen was then cooled and stored as in experiment 1. In both experiments, sperm motility parameters, plasma membrane integrity, and high mitochondrial membrane potential were assessed at 0, 24, and 48 hours post cooling. For experiment 3, 12 mares (n = 24 cycles) were bred with 48 hour–cooled semen from one stallion. Semen was processed as described in experiment 1. Mares had embryo flushing performed by 8-day post-ovulation. In experiment 1, BotuSemen Gold displayed superior total and progressive motility relative to INRA 96 (P < .05). There were no significant differences between the types of containers in any experiment. In experiment 2, INRA 96 and BotuSemen Gold extenders had similar total and progressive motility, but BotuSemen Gold had superior sperm velocity parameters at all timepoints. Embryo recovery was identical for both extenders (50%). Finally, the results obtained herein suggest that BotuSemen Gold is a suitable alternative to be included in semen cooling tests against INRA 96 in clinical practice.  相似文献   

18.
The aim of this work was to evaluate the effect of resveratrol (RSV) during liquid storage of stallion sperm for 24 hours at either 10°C or 4°C. The antioxidant RSV was added to reduce the oxidative damage that occurs during cold storage. Aliquots of 2 mL of diluted semen were stored either at 4°C or 10°C under anaerobic conditions, in the absence (control group) or presence of RSV at different concentrations (10, 20, 40, and 80 μM). Sperm quality parameters were assessed at 0 hours and after 24 hours of storage. Resveratrol treatment did not affect sperm quality parameters at 0 hours. At 24-hour storage, a significant (P < .01) decrease of sperm quality was observed independently from RSV supplementation and storage temperature. A significant decrease of viable spermatozoa with high mitochondrial membrane potential (SYBR+/PI−/JC-1+) was evident at 24-hour storage in 40- and 80-μM RSV groups compared with control group. Moreover, a decline of total motility in 80-μM RSV group compared with the control group and a decrease of progressive motility and average path velocity in 80-μM RSV group compared with control and 20-μM RSV groups were observed. In conclusion, our findings demonstrate that RSV supplementation does not enhance sperm quality of stallion semen after 24 hours of storage. Moreover, 40- and 80-μM RSV concentrations could damage sperm functional status, probably acting as pro-oxidant. Finally, although 24-hour storage significantly affected most of the sperm quality parameters, no significant differences were found in groups maintained at 4°C or 10°C, suggesting that stallion semen could be equally preserved at these different temperatures.  相似文献   

19.
This study aimed to assess the effects of sodium caseinate and cholesterol to extenders used for stallion semen cooling. Two ejaculates from 19 stallions were extended to 50 million/mL in four different extenders and cooled-stored for 24 hours at 5°C. The extender 1 (E1) consisted of a commercially available skim milk–based extender. The extender 2 (E2) consisted of E1 basic formula with the milk component being replaced by sodium caseinate (20 g/L). The extender 3 (E3) consisted of E1 basic formula added to cholesterol (1.5 mg/120 million sperm). The extender 4 (E4) consisted of a combination of the E2 added to cholesterol. At 24 hours after cooling, sperm motility parameters, plasma membrane stability (PMS), and mitochondrial membrane potential were assessed. In addition, cooled semen (1 billion sperm at 5°C/24 hours) from one “bad cooler” and one “good cooler” stallions, split into four extenders was used to inseminate 30 light breed mares (30 estrous cycles/extender). Milk-based extenders (E1 and E2) had superior sperm kinetics than E3 and E4 (P < .05). Plasma membrane stabilization was significantly higher (P < .05) in E4 than E1, whereas E2 and E3 presented intermediate values (P > .05). The mitochondrial potential intensity was lower (P < .05) in E2 and E4 groups compared with E1 and E3. The good cooler stallion had high fertility (∼80%) in all extenders. However, for bad cooler stallion, E1 40% (8/20) and E2 45% (9/20) had poor fertility (P < .05) compared with E4 85% (17/20), whereas E3 55% (11/20) had intermediate value (P > .05). In conclusion, the association of sodium caseinate and cholesterol improved fertility of bad cooler stallion semen cooled for 24 hours.  相似文献   

20.
Stallion semen cryopreservation is often associated with poor post-thaw sperm quality. Sugars act as nonpermeating cryoprotectants. The aim of the present study was to evaluate the cryoprotective effect of trehalose on stallion sperm quality and field fertility rates subjected to cooling and freeze–thaw process. Semen samples were collected from six Arabian stallions, divided into five different treatments in a final concentration of 100 × 106 sperm/mL by using INRA-82 extender containing 0, 25, 50, 100, and 200 mM of trehalose then subjected to both cold storage and cryopreservation. Sperm motility, acrosome, plasmatic membrane, and DNA integrity were analyzed, and 57 mares were used to evaluate the field fertility of chilled and frozen-thawed semen. Results showed that the extender containing 100 mM trehalose only increased the functional acrosomal, plasma membrane, and DNA integrities. The inclusion of 50 mM trehalose in semen extender resulted in significantly (P < .05) increased post-thaw total motility compared to the control group, and chilled semen achieved higher pregnancy rates compared to the frozen-thawed one. Pregnancy rate of mares inseminated with frozen-thawed semen (P < .05; 46.15% vs. 36.36%, respectively) was lower than those inseminated with chilled semen (76.47% vs. 68.75%, respectively) but higher than control. In conclusion, addition of 50 mM trehalose yielded the highest quality stallion semen after cooling and post-thawing in terms of motility, integrities of acrosome, membrane, and DNA as well as improved field fertility.  相似文献   

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