Effect of Centrifugation Technique on Post-storage Characteristics of Stallion Spermatozoa

Three ejaculates were collected from four stallions and used to compare the effects of three centrifugation methods on post-storage motility and recovery of available sperm. Two aliquots per ejaculate were diluted with skim milk-glucose (SKMG) extender to 50×10⁶ sperm/mL, placed in 50-mL conical bottom tubes, and centrifuged at either 700g for 15 minutes (700g) or 600g for 12 minutes (600g). A third aliquot was diluted 1:1 with SKMG, placed in 15-mL conical tubes, and centrifuged at 400g for 7 minutes (400g). Subsamples from each pre-treated diluted ejaculate were held at room temperature and evaluated for motility at the same time as the post-centrifugation pre-storage motility evaluation was made for treated aliquots. After centrifugation, samples from each aliquot were stored at 5°C for evaluation after 24 and 48 hours or frozen in liquid nitrogen. Percentage of available sperm harvested was higher (P ≤ .05) for aliquots centrifuged in 15-mL tubes at 400g versus 600g in 50-mL tubes. After centrifugation, total but not progressive motility of aliquots centrifuged at 700g was lower than that for noncentrifuged controls and sperm from aliquots centrifuged at 400g in 15-mL tubes. After cold storage, values for total but not progressive motility or velocity were higher (P ≤ .05) for aliquots centrifuged in 15-mL tubes at 400g compared with those centrifuged in 50-mL tubes at both 600g and 700g. Postthaw motility of frozen sperm was not different between centrifugation treatments. Poststorage percentages of intact acrosomes and detached heads did not differ because of centrifugation treatment.     

Effects of Egg Yolk Source on the Cryopreservation of Stallion Spermatozoa

Three experiments were conducted to determine whether replacement of chicken egg yolk, as a component of freezing extenders, with egg yolk from other avian species would improve the post-thaw motility and percentage of intact acrosomes of stallion spermatozoa. In the first experiment, substitution of chicken egg yolk with chukar egg yolk, as a component of the lactose-ethylenediaminetetraacetic acid extender, improved (P ≤ .05) the post-thaw motility of stallion spermatozoa. These results were not replicated in (IMV Technologies, Maple Grove, MN, USA) a more expansive study comparing 2%, 4%, 6%, or 8% egg yolk combined with INRA 96 when a “slow freeze” method was used, or the same substitution at levels ranging from 13% to 22% when egg yolk was combined with lactose-ethylenediaminetetraacetic acid for diluents used for a “fast freeze” method of cryopreservation. In the third study, egg yolks from regular and high omega-3 chicken eggs as well as from turkey, chukar, and mallard duck eggs were analyzed for lipid content and fatty acid profile. The yolk from the turkey eggs was higher (1,300 mg/100 g) and that from mallard ducks was lower (560 mg/100 g) in cholesterol as compared with the two types of chicken eggs and chukar egg yolk (range, 1,046-1,094 mg/100 g). In addition, the high omega-3 eggs did test higher for fatty acids (4.51 g/100 g) than other types of eggs (range, 0.28-0.73 g/100 g). Substitution of chicken egg yolk with turkey, but not duck, egg yolk resulted in higher post-thaw total motility (P ≤ .05) for spermatozoa obtained from two of the three stallions used in the third experiment.     

Comparison of Various Extenders for Storage of Cooled Stallion Spermatozoa for 72 Hours

Multiple extenders have been developed to preserve cooled stallion semen. Comparisons of some extenders have been made but there is need for further research in this area. Extenders tested included EZ Mixin (Animal Reproduction Systems, Chino, CA), Kenney's, Universal (NASCO, Fort Atkinson, WI), EquiPro, EquiPro CellGuard (Minitube of America, Verona, WI), and INRA 96 (IMV, Maple Grove, MN). Semen was collected and each ejaculate was divided and extended in each of the aforementioned extenders and stored at 4°C. Motility measures were determined using computer-assisted sperm analysis at 0, 24, 48, and 72 hours after collection. Samples were evaluated for total motility, progressive motility (PM), straight-line velocity, curvilinear velocity, straight-line distance, and curvilinear distance. Total motility and PM decreased over time in storage (P < .05). Sperm stored in INRA 96, EquiPro, and EquiPro Cell Guard retained the most total motility and PM over the 72 hour period (P < .05). Universal, EquiPro, and EquiPro Cell Guard had the highest measurements for curvilinear velocity, straight-line velocity, and curvilinear distance (P < .05). There were no significant differences among the extenders for straight-line distance.     

Motility and Fertility Evaluation of Thawed Frozen Stallion Semen After 24 Hours of Cooled Storage

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.     

Cryopreservation of Stallion Spermatozoa with INRA96 and Glycerol

The aim of the present study was to improve success of cryopreservation of stallion spermatozoa. Semen from eleven stallions was collected and frozen in INRA 96 with two different concentrations of glycerol (3.5% and 6.0%) and compared with a control freezing process. The mean post-thaw motility for the eleven stallions of 57.93% (3.5% glycerol) and 66.50% (6.0% glycerol), which was statistically higher (P < 0.05) when compared with the mean post-thaw motility (39.7%) for semen in a control egg-yolk extender (Equipro^® CryoGuard™ Complete, Minitube). The Equipro^® CryoGuard™ Complete is a commercial semen freezing protocol that has been one of the standard processes used in our laboratory for freezing equine spermatozoa. INRA 96 with 6% added glycerol was used in the fertility trial as it provided the highest spermatozoa survival. To evaluate fertility of the frozen semen, eight mares were bred over two cycles with both fresh and frozen semen. The pregnancy rate of mares bred with frozen semen (55.6%) was not statistically different (P > 0.05) from the pregnancy rate of mares bred with fresh semen (55.6%). INRA 96 with 6.0% glycerol improved the survivability of stallion spermatozoa through the cryopreservation process, and subsequent fertility was not different (P > 0.05) from fresh, extended semen.     

Nicotinic Acid (Niacin) Supplementation in Cooling and Freezing Extenders Enhances Stallion Semen Characteristics

In the present study, we aimed to evaluate the possible protective effects of the nicotinic acid (NA) at three concentrations (10, 20, and 40 mM) on the equine cooled and frozen-thawed spermatozoa quality markers including viability, plasma membrane or acrosome integrity, DNA fragmentation, lipid peroxidation, and total oxidant levels. We also evaluated the effects of NA on preservation of the post-thaw sperm quality after 6 hours of cold storage before freezing. Five stallions were used for semen collections. The current experiment was repeated six times using pooled semen samples from two stallions, each time. We showed that NA at 20 and 40 mM concentrations could significantly improve the stallion sperm quality markers during cold storage. However, the protective effects were not different between 20 mM and 40 mM concentrations in most measures. Nicotinic acid could also improve the post-thaw stallion sperm quality at 10, 20, and 40 mM concentrations. However, the 40 mM concentration showed a negative impact on some post-thaw kinematic sperm parameters. Nicotinic acid at 10 and 20 mM concentrations could preserve the sperm cryo-tolerance to be frozen up to 8 hours after collection without a significant decline in most of the post-thaw sperm quality measures. Nicotinic acid could also decrease the level of the lipid peroxidation and total reactive oxygen/nitrogen species in the cooled and frozen-thawed spermatozoa, in a dose-dependent manner. Therefore, NA at 20 mM concentration could preserve most of the stallion sperm quality measures during cold storage (42 hours, 5°C) and enabled storage of cooled stallion semen for 6 hours before freezing without significant deterioration of the post-thaw sperm quality.     

Stallion Semen Cooling Using Native Phosphocaseinate-based Extender and Sodium Caseinate Cholesterol-loaded Cyclodextrin-based Extender

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.     

Studies on Motility and Fertility of Cooled Stallion Spermatozoa

This study on extended, cooled stallion spermatozoa aimed to compare the ability of three extenders to maintain sperm motility during 24 h of preservation, and to describe pregnancy and foaling rates after artificial insemination (AI) of stallion spermatozoa stored and transported in the extender chosen from the in vitro study. After 6 and 24 h of preservation, motility, both subjective and evaluated by the motility analyzer (total, progressive and rapid), was lower in non-fat, dried skim milk-glucose than in both other extenders: dried skim milk-glucose added to 2% centrifuged egg yolk, and ultra high temperature treated skim milk-sugar-saline solution added to 2% centrifuged egg yolk (INRA82-Y). Rapid spermatozoa and sperm velocity parameters, after 24 h, were significantly higher in INRA82-Y. In the fertility trial, semen collected from three Maremmano stallions, diluted in INRA82-Y, and transported in a refrigerated Styrofoam box, was used to inseminate 56 mares of the same breed. Pregnancy rates after the first cycle and per breeding season were significantly higher for the 31 mares inseminated in three AI centres (54.8 and 80.6%, respectively) than for the 25 mares inseminated at the breeder's facilities (28.0 and 52.0%). Foaling rates were not significantly different between the AI centres mares (54.8%) and the other mares (44.0%). In conclusion, INRA82-Y yielded satisfactory pregnancy and foaling rates, especially when employed in the more controlled situation of an AI centre, and can therefore be included among those available for cooled stallion semen preservation.     

The Use of Cefquinome in Equine Semen Extender

Antibiotics are commonly used in equine semen extender for conservation, if semen has to be stored cooled for a maximum of 48 hours or frozen, to eliminate pathogenic or potentially pathogenic bacteria from semen and reduce the risk of postmating endometritis. Little is known about the effect of antibiotics on spermatozoa when semen is stored over a longer period. Cefquinome, a broad spectrum antibiotic and fourth-generation cephalosporin, has been proven to be a powerful drug for the treatment of endometritis and mastitis in different species. Recently in equine studies, it was found to localize in high concentrations in the endometrium. Therefore, cefquinome was used as the antibiotic in semen extender and compared with a commercial semen extender containing gentamicin for effects on motility and membrane integrity of spermatozoa. During the breeding season, ejaculates from nine light horse stallions were collected and half of each ejaculate was stored for 48 hours in modified Kenney type semen extender containing either cefquinome or gentamicin. At 0, 24, and 48 hours, aliquots (20 μL) of the stored semen were evaluated for (progressive) motility and membrane integrity, as well as for various motility parameters by computer assisted sperm analysis. No differences (P > .05) were found in total motility or progressive motility between extenders at any time point. However, there were differences (P < .05) in velocity parameters, although the effect of velocity parameters on fertility is not clear. In general, semen parameters after storage in non-fat dried skim milk semen extender containing cefquinome are comparable with those after storage in semen extender containing gentamicin. The wider spectrum of bactericidal activity possessed by cefquinome may prove to be beneficial in some cases.     

Effect of BAPTA-AM on Thawed Stallion Spermatozoa Extended in INRA 96 or Tyrode's Medium

We studied the effect of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) on the outcome of cryopreservation of stallion spermatozoa and whether reextension of thawed sperm in a more physiological and Ca²⁺-containing medium might improve the characteristics of thawed stallion spermatozoa. Individual ejaculates from six stallions were collected and split into three subsamples. The first two samples were supplemented with the membrane-permeable Ca²⁺ chelator BAPTA-AM at final concentrations of 5 and 10 μM, respectively, while the third subsample served as control. After 4 weeks of storage, samples were thawed in a water bath at 37°C and evaluated using flow cytometry and computer-assisted sperm analysis (CASA). In a second experiment, in order to determine whether restoring Ca²⁺ could improve sperm quality after cryopreservation, thawed semen was washed by centrifugation and resuspended in Tyrode's complete medium. BAPTA-AM supplementation did not modify the outcome of cryopreservation; however, changing the spermatozoa from INRA 96 to Tyrode's complete medium resulted in significant improvements in the percentages of live sperm and total motility post thaw.     

The Cryoprotective Effect of Trehalose Supplementation on Boar Spermatozoa Quality

In order to improve boar sperm quality during frozen-thawed process, the influence of the presence of trehalose on success of cryopreservation of boar sperm were investigated. We evaluated freeze-thawing tolerance of boar spermatozoa in a base cooling extender with the addition of different trehalose concentrations (0, 25, 50, 100 and 200 m m ), and try to determine the optimum concentration of trehalose. We chose sperm motility, mitochondrial activity, acrosome integrity and membrane integrity as parameters to evaluate cryopreservation capacity of boar spermatozoa. We obtained the best results for 100 m m trehalose-supplemented extenders, with values of 49.89% for motility, 44.69% for mitochondrial activity, 66.52% for acrosome integrity and 44.61% for membrane integrity, while freeze-thawing tolerance diminished significantly for 200 . The synergic effect of trehalose and glycerol resulted in better cryosurvival of boar spermatozoa than that of a single cryoprotectant. In conclusion, when trehalose-supplementation was added up to 100 m m , trehalose confers a greater cryoprotective capacity to the extender, and the sperm motility, mitochondrial activity, membrane integrity and acrosome integrity parameters were significantly improved during frozen-thawed process.     

Effect of Seminal Plasma Components on the Quality of Fresh and Cryopreserved Stallion Semen

The importance of seminal plasma (SP) components for stallion semen quality and freezability is little known. This study aimed to evaluate the relationship between SP components and fresh/cryopreserved stallion semen quality. Semen of 30 stallions was collected, and then, SP was recovered and lyophilized. Total protein (TP), vitamin C (CVIT), vitamin E (EVIT), vitamin A (AVIT), iron (Fe), copper (Cu), magnesium, and zinc (Zn) in SP were assessed. Sperm was frozen in an extender supplemented with lyophilized SP. In fresh semen motility, abnormal morphology (AM), sperm vitality (SV), and plasma membrane integrity (PMI) were evaluated. In post-thaw semen, additionally, total motility (TM), progressive motility (PM), straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat cross-frequency (BCF) were assessed. Levels of component of SP were established by a distribution analysis. Generalized linear models were fitted. Comparisons of means were done with Tukey's test. Correlation and regression analyses were performed. Vitamins and ions were found to be related to fresh semen quality. For post-thaw sperm, medium TP showed higher semen quality. Negative regression and correlation coefficients between CVIT and all post-thaw semen parameters were found. Low EVIT yielded the lowest PM, VSL, and VAP values, while a high level of AVIT yielded the best results for sperm quality. A high level of Cu yielded higher results for TM, PM, VCL, and ALH. Moreover, a negative correlation was found between Zn, SV, and PMI. In conclusion, SP composition influences fresh and post-thaw stallion semen quality.     

Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid

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.     

Case Study of Processing and Insemination Techniques: Attempts to Improve Fertility of an Aged Stallion with Dilute Semen of Poor Quality

The objective of this case study was to investigate whether semen centrifugation and low-dose insemination techniques would improve fertility of an aged subfertile Quarter Horse stallion with low sperm concentration, motility, and morphology in ejaculates. Forty-five mares were bred by one of five treatments (n = 9 per group) using the entire ejaculate as follows: (1) Group Body: body insemination with ejaculate diluted 1:1 in TAMU extender; (2) Group Body-Cent: body insemination after centrifugation and re-suspension of sperm pellet to 1 mL in TAMU extender; (3) Group Horn-Cent: deep horn insemination after centrifugation and re-suspension of sperm pellet to 1 mL in TAMU extender; (4) Group Cent-Hys: hysteroscopic insemination onto the uterotubal papilla after centrifugation and re-suspension of sperm pellet to 200 μL in Kenney-Modified Tyrode’s extender; and (5) Group Dens-Hys: hysteroscopic insemination onto the uterotubal papilla after discontinuous density gradient centrifugation and re-suspension of the sperm pellet in 200-μL Kenney-Modified Tyrode’s extender. Pregnancy rates did not differ among treatment groups (P = .77). Semen centrifugation for low dose insemination did not appear to improve fertility of this subfertile stallion, despite use of entire ejaculates for each individual insemination dose.     

Effects of Hyaluronan Supplementation on Cryopreserved Equine Spermatozoa Hyaluronan and Cryopreserved Equine Spermatozoa

Addition of hyaluronan, a nonsulfated glycosaminoglycan, to fresh and frozen thawed human semen results in substantial retention of motility over time. Hyaluronan also has been reported to preserve postthaw viability and maintain membrane stability of boar spermatozoa. Therefore, experiments were designed to investigate the use of a commercially available hyaluronan (Map-5, Bioniche Animal Health, Inc., Athens, GA) in freezing extender for cryopreservation of equine spermatozoa. In experiment 1, aliquots from ejaculates were supplemented before freezing with one of four levels of hyaluronan: 100 μg/mL, 200 μg/mL, 400 μg/mL, and 1000 μg/mL along with an untreated control. No differences in sperm motility, assessed by computer-assisted sperm motility analysis (CASA), were found for any treatment at times 0, 30, or 60 minutes postthaw. Decreases in motility were noted in the highest hyaluronan group (1,000 μg/mL) after 90 and 120 minutes of incubation. Sperm viability, as assessed using SYBR-14/propidium iodide staining, was decreased (P < .05) when treated with 1,000 μg/mL compared with the control (37.1% and 46.1%, respectively). Motility parameters tended to remain elevated in those ejaculates treated with 200 μg/mL at various time points. Experiment 2, therefore, further investigated the effects of hyaluronan at 200 μg/mL on motility parameters and acrosome integrity and zona pellucida binding. Total (TM) and progressive (PM) motility of treated sperm immediately after thawing and at 60 minutes post-thaw were higher compared with control (P < .05). A tendency (P < .1) to maintain TM at 90 and 120 minutes post-thaw also was noted. No differences were noted for the mean number of spermatozoa bound to bovine oocytes for control or treated sperm (22 ± 14 vs 25 ± 17, respectively). Acrosome integrity also was unchanged between the two groups based on fluorescein isothiocyanate (FITC)−peanut agglutinin (PNA)/propidium iodide staining. All samples contained <1% live acrosome-damaged spermatozoa. In the final experiment, the effects of hyaluronan supplementation post-thaw was investigated using hyaluronan concentrations of 100, 200, and 400 μg/mL. Motility parameters studied over an 8-hour period at 37°C yielded no consistent differences. In conclusion, addition of hyaluronan at a concentration of 200 μg/mL before freezing increased spermatozoal post-thaw motility. High concentration of hyaluronan (1,000 μg/mL) appeared to be detrimental to post-thaw motility. Effects of hyaluronan on fertility are beyond the scope of this study and have yet to be determined.     

Effects of Repeated Partial Thaw and Refreeze on Post-Thaw Parameters of Stallion Semen Cryopreserved in Cryovials

The current study evaluated post-thaw semen parameters of stallion semen cryopreserved in cryovials and subjected to multiple partial thaw-refreeze cycles. Five fertile stallions were collected twice, and ejaculates were analyzed for concentration, percent membrane integrity, motility, morphology, and sperm chromatin structure (SCSA). Semen processed with freezing extender from each ejaculate was cryopreserved in both 1.2-mL cryovials and 0.5-mL straws. Cryovials were subjected to eight subsequent partial thaw-refreeze cycles. Cryovials were warmed for approximately 30 seconds; then, a sample of cryopreserved semen was removed with a 16-gauge needle, and the cryovial was immediately refrozen in liquid nitrogen. A piece of 0.5-mL straw cut under liquid nitrogen from the same stallion and ejaculate was thawed alongside each cryovial to serve as a control. Thawed samples were analyzed for percent membrane integrity, motility, and SCSA. Post-thaw parameters of motility and membrane integrity were analyzed by one-way or two-way analysis of variance with repeated measures when appropriate. The SCSA data were analyzed using a mixed regression model. Post-thaw motility and percentage of intact sperm were significantly lower when sperm was cryopreserved in cryovials compared to straws. However, these parameters may remain adequate for use in assisted reproductive techniques (ARTs) such as intracytoplasmic sperm injection through all cryovial thaws. Additionally, DNA denaturability was not affected by semen packaging method and was only affected by thaw number, increasing at post-thaws 5 and 6. This technique may offer a unique approach for cryopreservation and utilization of stallion sperm for ARTs in the future.     

甘油和海藻糖对低温保存精液品质的影响

为了研究低温保存下海藻糖和甘油对牛精液品质的影响,本试验选取了宁夏四正生物技术公司的健康荷斯坦种公牛的精液,在低温保存液里分别添加5%的甘油以及0.028mol/L、0.014mol/L、0.0416mol/L的精液保护剂海藻糖为试验组,以不添加试剂的低温保存精液为对照组,对保存不同时间的精液品质进行测定。结果显示添加甘油的试验组精子活力低于对照组,但差异不显著（P〉0.05）。海藻糖浓度为0.0416mol/L组的精子畸形率低于对照组及其他海藻糖组,并且差异显著（P〈0.05）;海藻糖组与对照组精子活力相比,在24h、48h、72h时差异不显著（P〉0.05）;在96h、216h、240h时差异显著（P〈0.05）,其活力下降明显。     

Dietary Polyunsaturated Fatty Acid Supplementation Improves the Quality of Stallion Cryopreserved Semen

The objective was to assess the influence of polyunsaturated fatty acid supplementation on the quality of fresh, cooled, and frozen-thawed stallion semen. Ten stallions received their normal diet (control group) or normal diet plus 150 mL of polyunsaturated fatty acid (PUFA) linseed-based oil (PUFA group). Semen was collected every 15 days during 60 days. Stallions were reversed across the treatments after a sixty-day interval. Semen was evaluated at 2, 6, 12, and 24 hours after cooling and 24 hours after freezing. Motility (MOT), vigor, membrane viability, morphology, acrosome integrity, and osmotic tolerance test (OTT) were evaluated. In the frozen-thawed semen, sperm dynamic characteristics were analyzed by computer-assisted sperm analysis and thiobarbituric acid reactive substances (TBARs) determined. The effects of treatment, time, semen type, and their interactions were submitted to PROCMIX (SAS) and means compared by the Tukey test. There was no treatment effect on the quality of fresh and cooled semen. However, frozen-thawed semen MOT, vigor, and OTT were superior (P < .05) in control compared to PUFA group. An interactive effect of sample day by treatment was observed, such that, TBARs increased over time (P = .002) in the PUFA group after 15, 30, 45, and 60 days from the beginning of supplementation. Thus, sperm may become more susceptible to the reactive oxygen species, probably due to the incorporation of polyunsaturated fat in the cell membrane. The addition of PUFA-enriched oil may be an alternative for improving frozen-thawed semen quality by increasing its MOT and resistance to osmotic changes to which sperm cells are submitted during the freezing process.     

Positive Effect of Manganese (III) Meso-Tetrakis (4-Benzoic Acid) Porphyrin on Stallion Spermatozoa During Storage in Cool Condition

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.