Skim Milk-based Extender Research Articles

Pre- and Post-Thaw Addition of L-Carnitine and Pyruvate: Effect on Stallion Sperm Parameters.

The addition of antioxidants to cryopreservation media reportedly improves sperm post-thaw quality and reproductive performance after artificial insemination. Therefore, the objectives of this study were to evaluate if the addition of L-carnitine and pyruvate to freezing media, or their addition to samples after thawing, improves the post-thaw quality of equine spermatozoa. Thus, in Experiment 1, stallion semen samples were cryopreserved in: (1) EDTA-glucose-based extender with 20% egg yolk and 5% dimethylformamide (EDTA control); (2) skim milk-based extender with 20% egg yolk and 5% dimethylformamide (milk control); (3) Extender1 supplemented with 50 mM L-carnitine and 10 mM pyruvate (EDTA-carnitine-pyruvate); and (4) Extender 2 supplemented with 50 mM L-carnitine and 10 mM pyruvate (milk-carnitine-pyruvate). In Experiment 2, 50 mM L-carnitine and 10 mM pyruvate were added post-thaw to samples cryopreserved with extenders 1 and 2 (EDTA control and milk control). Sperm kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability were evaluated after thawing. No significant differences (p > 0.05) were observed for most of the kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability of spermatozoa, between the samples frozen in the presence or absence of L-carnitine and pyruvate, nor between the samples after the post-thaw addition of these components. A higher (p < 0.05) mean velocity and higher (p < 0.05) amplitude of lateral head displacement were observed in the samples frozen in the milk-based extender with the addition of L-carnitine and pyruvate after thawing. The addition of 50 mM L-carnitine and 10 mM pyruvate, either to the freezing extenders or after thawing, was not deleterious for sperm; however, it did not improve equine sperm motility, viability, acrosome and DNA integrity, nor decrease membrane lipid peroxidation after thawing.

Effects of Origanum majorana essential oil and antibiotics on the quality of frozen thawed Beni Arouss buck semen.

This study aims to investigate the effects of Origanum majorana (OM) essential oil (EO) at different concentrations and antibiotics on post-thawed Beni Arouss buck semen quality. Semen collection was performed using artificial vagina from eight Beni Arouss bucks. Ejaculates were pooled, divided into 12 equal aliquots, washed and diluted to 400 × 106 sperm/ml (with 7% of glycerol). Skim milk-based extender was supplemented with different concentrations of OM EO (0%; 0.01%; 0.02%; 0.03%; 0.04% and 0.05%) without antibiotics, marked as (CTR-), (M1-), (M2-), (M3-), (M4-) and (M5-), and with 50 mg of streptomycin and 50,000 IU of penicillin per 100 ml, marked as (CTR+), (M1+), (M2+), (M3+), (M4+) and (M5+), respectively. Aliquots were cooled to 4°C, then frozen in 0.25 ml straws with a programmable freezer and finally stored in liquid nitrogen for 48 h. Thawing was performed at 37°C for 30 s. Motility, live sperm, sperm abnormalities, membrane integrity, lipid peroxidation and bacterial growth were evaluated after thawing. Among the tested extenders, M2+ improved all semen quality parameters. Sperm motility, live sperm and membrane integrity increased significantly, while the number of abnormal sperm and bacterial growth decreased significantly. The toxic effect of OM EO, with and without antibiotics, appeared beyond 0.03%. In conclusion, M2+ is recommended to improve the cryopreservation of Beni Arouss buck semen.

Effect of ejaculation frequency on ram semen characteristics, seminal plasma composition and chilled sperm quality

The aim of this study was to see how chilled sperm quality is affected by ejaculation frequency and its correlation to seminal plasma composition in INRA 180 rams. Five rams were collected at high (HFE) and low frequencies (LFE). For the high frequency, the rams were collected three times on the same day every three days for 18 days. In the low frequency collection, three consecutive ejaculates were collected once a week for four weeks. Ejaculates were collected at 20-minute intervals in either HFE or LFE. Semen characteristics, concentration of total protein, lipid, cholesterol and fructose in seminal plasma were assessed. Semen samples were extended in skim milk-based extender at 15 °C, then evaluated at different storage times (0, 8, and 24 h). Fresh sperm quality parameters, seminal plasma composition, and stored sperm quality were shown to be higher in LFE than in HFE and in the first and second ejaculates than in the third one. After 24 h of storage, sperm quality was correlated to seminal plasma components. In conclusion, the frequency of ejaculation has an effect on the fresh and stored semen quality as well as on seminal plasma composition in INRA 180 rams.

Cholesterol-Loaded Cyclodextrin Addition to Skim Milk-Based Extender Enhances Donkey Semen Cooling and Fertility in Horse Mares

The present study aimed to compare semen parameters and fertility of cooled donkey semen extended in a commercially available skim milk (SKM) based extender and the same extender with cholesterol-loaded cyclodextrin (SKM-CLC). In Experiment 1, thirty-five ejaculates from seven jacks were split in SKM and SKM-CLC, extended at 50 million sperm/mL and stored at 5°C for 48 hours. Total motility (TM), progressive motility (PM), percentage of sperm with rapid motility (RAP) were assessed with CASA. Plasma membrane stability (PMS), and high mitochondrial membrane potential (HMP) were assessed with the combination of Yo-Pro and MitoStatusRed with flow cytometry. Semen was assessed before (0), 24 and 48h after cooling. In Experiment 2, two estrous cycles of 15 mares were used for fertility assessment. Mares were examined every other day by transrectal ultrasonography and had ovulation induced with 250 µg of histrelin acetate when a ≥35 mm follicle was first detected. Mares were randomly inseminated with semen obtained from one jack. Semen was extended in either SKM or SKM-CLC and cooled-stored for 24 hours. Pregnancy diagnosis was carried out 15-day post-ovulation. Data were analyzed with a mix model and Tukey's as posthoc and logistic regression model. Significance was set at P ≤ .05. There were no differences in TM, PM, RAP, PMS, and HMP for semen extended in either extender immediately before cooling (P > .05). There was a reduction in TM, PM, RAP, PMS, and HMP overtime across groups (P < .05); however, semen extended with SKM-CLC had superior TM, PM, RAP, PMS, and HMP than semen extended in SKM at 24- and 48-hours post-cooling (P < .05). Mares bred with semen extended in SKM had a lower conception rate (13%, 2/15 cycles) than cycles bred with SKM-CLC (47%, 7/15 cycles; P < .05). In conclusion, incorporating CLC into SKM extender improved cooling ability and fertility of donkey semen in horse mares. It remains to be determined if similar results can be obtained in clinical practice with mares and jennies.

Donkey Epididymal Transport for Semen Cooling and Freezing.

Simple SummaryIn the event of death, euthanasia, or forceful castration for medical reasons, epididymal semen harvesting represents the last opportunity to preserve the genetic material of valuable sires. However, this technique has yet to be tested in donkeys. Three experiments were carried out to assess epididymal semen cooling and freezing in donkeys. In experiment 1, semen cooling and freezing were conducted immediately after castration, and in experiments 2 and 3, epididymides were shipped overnight, and then epididymal semen cooled and frozen. Results showed that cooling of epididymal semen up to 24 h after harvesting did not affect motility parameters or plasma membrane integrity. Collectively, the post-thaw results revealed low motility parameters across groups; At the same time, the plasma membrane integrity did not reflect this trend, and the values remained high, suggesting that there was a lack of epididymal sperm activation after freezing. In summary, freshly harvested and cooled-shipped epididymal donkey semen had satisfactory semen parameters. New studies need to address donkey epididymal semen fertility in mares and jennies.The objectives of this study were to assess the cooling and freezing of donkey epididymal semen harvested immediately after castration (Experiment 1, n = 4) or after the shipment (24 or 48 h) of epididymides attached to testicles (Experiment 2, n = 14) or dissected apart (Experiment 3, n = 36). In each experiment, semen was frozen immediately (Non-Centrif) in an egg yolk-based semen extender (EY) or after processing through cushion-centrifugation (Centrif) while extended in a skim milk-based extender (SC). In all three experiments, cooled, pre-freeze, and post-thaw epididymal semen was assessed for total motility (TM), progressive motility (PM), plasma membrane integrity (PMI), and high mitochondrial membrane potential (HMMP). Data were analyzed with R using mixed models and Tukey’s test as posthoc. Results showed that the cooling of epididymal semen up to 24 h after harvesting did not affect motility parameters or plasma membrane integrity; furthermore, in Experiment 3, the post-thaw evaluation of both Centrif and Non-Centrif achieved similar TM and PM. Collectively, the post-thaw results revealed low motility parameters across groups; while, the PMI and HMMP did not reflect this trend, and the values remained high, suggesting that there was a lack of epididymal sperm activation with either centrifugation or extenders. In summary, freshly harvested and cooled-shipped and cooled semen had satisfactory semen parameters. Future studies need to address donkey epididymal semen fertility in mares and jennies.

Effect of quercetin on the motility of cryopreserved canine spermatozoa

The addition of an antioxidant to cryopreservation solutions for preventing oxidative stress to sperm from several species, including that from humans, has been studied previously. Quercetin is a flavonoid contained in subarctic trees with freeze resistance and is known to be a strong antioxidant. Therefore, the effect of quercetin on the cryopreservation of dog spermatozoa was examined in this study. The proportions of total motile spermatozoa were significantly higher at 30, 60, 90, 120, and 150 min and at 60, 120, and 150 min after thawing in groups treated with 5 μg/ml and 10 μg/ml of quercetin dissolved in 0.1% DMSO added to the second extender based on skim milk compared to that in the control group, respectively. There were no differences between the experimental groups in the proportion of total motile spermatozoa during the observation periods. The proportion of total motile spermatozoa among those treated with 5 μg/ml of quercetin in 0.1% DMSO was improved by approximately 10–20% at 30–180 min after thawing compared to that in the control group. To evaluate the fertility of cryopreserved spermatozoa treated with quercetin, 2 × 108 spermatozoa were transcervically inseminated into bitches, and a total of 18 puppies were delivered in three bitches. These results indicated that supplementation of quercetin as a cryoprotectant to the skim milk-based extender improved the motility of cryopreserved spermatozoa from dogs compared to those of the control group. And fertility of cryopreserved spermatozoa with quercetin supplementation was proven with higher efficiency.

Intracellular calcium chelating agent (BAPTA-AM) aids stallion semen cooling and freezing-thawing.

This study aimed to investigate the effects of different concentrations of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N0N0-tetraacetic acid, tetra-acetoxymethyl ester (BAPTA-AM), an intracellular calcium chelating agent, on stallion semen cooling and freezing-thawing. After collection, semen was extended (1:1 v/v) on a skim milk-based extender, centrifuged and resuspended at 400million/ml into cooling or freezing extenders containing 0, 5, 25, 50, 100 and 200μΜ BAPTA-AM. Motility parameters were assessed after cooling in Equitainer at 5°C for 12, 24, 48, 72 and 120hr and after freezing-thawing. In addition, mitochondrial membrane potential, intracellular ATP, reactive oxygen species and malondialdehyde concentrations were measured in cryopreserved-thawed semen. Cooled stored (48hr) semen containing 50μΜ BAPTA-AM and control extender (0μΜ BAPTA-AM) was used to assess fertility. Inclusion of 50μΜ BAPTA-AM resulted in superior sperm motility parameters during cooled storage when compared to other groups (p<0.05). Furthermore, semen cryopreserved in extender containing 50μΜ BAPTA-AM showed increased intracellular ATP and mitochondrial membrane potential, whereas reactive oxygen species and malondialdehyde were increased after thawing for all groups (p<0.05). Addition of 50μΜ BAPTA-AM to cooling extender resulted in similar pregnancy rates to the control group (75% vs. 73.6%, respectively; p>0.05). In conclusion, the addition of BAPTA-AM to semen extenders aided stallion semen cryopreservation in a dose-dependent manner. Furthermore, the cooling extender supplemented with 50μΜ BAPTA-AM could be used to prolong the sperm motility during cooling without apparently compromising fertility. Field trials should be conducted to assess fertility of cryopreserved stallion semen with BAPTA-AM.

The Effect of Four Different Commercial Semen Extenders on the Motility of Stallion Epididymal Sperm

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.

Novel technical strategies to optimize cryopreservation of goat semen using cholesterol-loaded cyclodextrin

Artificial insemination is well-established in dairy cattle, with sires housed in commercial studs for processing. In some species, however, sires located on-farm are used for artificial insemination by shipping their semen to an off-site laboratory for processing within 24 h of collection. To expedite semen transport from the farm to laboratory, protocols must be uncomplicated. For goat semen, an obstacle is the seminal plasma, which must be removed because it contains proteins that impede sperm quality. Our objective is to develop a simple strategy to transiently store goat semen for 24 h prior to freezing. Cholesterol-loaded cyclodextrin (CLC) has been demonstrated to improve sperm tolerance to cryopreservation. Therefore, we hypothesized that CLC improves goat sperm resistance to seminal plasma damage, over 24 h prior to cryopreservation. We first evaluated the ability of CLC to protect goat sperm against seminal plasma damage by treating fresh semen with or without seminal plasma prior to cryopreservation. Second, fresh goat semen with seminal plasma was extended in skim milk-based extender ± CLC and held for 24 h at 5 °C prior to freezing. Our results indicate that CLC treatment improves goat sperm resistance to seminal plasma-mediated injury and protects sperm quality over 24 h prior to freezing (P < 0.05). Although the in vivo fertility of semen must first be assessed, it is possible that protocols for goat semen cryopreservation can be simplified by including CLC and eliminating seminal plasma removal. Processing and distribution of goat semen for AI would thereby be facilitated.

Cholesterol-loaded cyclodextrin improves ram sperm cryoresistance in skim milk-extender

Cholesterol-loaded cyclodextrin (CLC) is known to improve ram sperm cryosurvival. This study expands on previous research to: (1) determine the mechanism by which CLC improves ram sperm cryosurvival and (2) compare the efficiency of a novel, skim milk-based extender containing CLC to a traditional egg yolk-based extender. Hypothesis #1 was that CLC enhances membrane cholesterol content to increase the resistance of ram sperm to cold and osmotic stress, thereby improving cryosurvival. We first assessed the ability of fresh sperm treated with CLC to withstand cold shock. Second, fresh sperm were treated with CLC to evaluate their tolerance to osmotic stress. Third, to confirm that cholesterol is incorporated into the sperm using CLC, we quantified sperm cholesterol. To test Hypothesis #2 that CLC is most effective in a medium without competing cholesterol, we compared sperm cryosurvival and fertility in skim milk-based extender containing CLC versus in a traditional egg yolk-based freezing extender without CLC. Our data confirmed that CLC treatment improves ram sperm cold shock and osmotic stress resistance, and augments sperm cholesterol content. Semen in skim milk-based extender containing CLC prior to freezing, had more motile sperm with intact acrosomes after thawing compared to semen in egg yolk-based extender. In contrast, sperm plasma membrane integrity and in vivo fertility of the semen cryopreserved in the skim milk-based extender with CLC did not differ from semen that was cryopreserved in egg yolk-based extender. Further research is warranted to combine CLC with other cryoprotection strategies or to modify the insemination protocol.

66 EFFECT OF ADDITION OF CHOLESTEROL-LOADED CYCLODEXTRIN BEFORE FREEZING ON QUALITY AND FERTILITY OF STALLION FROZEN SEMEN

The aim of the present study was to evaluate the effect of cholesterol-loaded cyclodextrin on the quality and fertility of stallion frozen semen. Three ejaculates from each of 4 stallions were used. The semen was diluted (1 : 1) with a skim milk-based extender (Botu-SemenTM, Botupharma, Brazil). The samples were divided into 2 groups: control group (CG), composed of semen diluted only with extender, and treated group (TG), composed of semen diluted with extender plus 750 mg mL–1 of cholesterol-loaded cyclodextrin. Both groups were incubated for 15 min at 20°C. The semen was frozen with Botu-CryoTM (Botupharma, Brazil) extender according to the manufacturer's protocol in 0.5-mL straws containing 100 × 106 of total sperm. The sperm kinetic parameters were analysed by computer-assisted semen analysis, and plasma membrane integrity by flow cytometer (propidium iodide and fluorescein isothiocyanate -PSA) on post-thaw. The fertility trial was carried out inseminating 2 cycles of 20 mares (total of 40 cycles) immediately post-ovulation using 4 straws of CG or TG (400 × 106 total sperm), one from each stallion in a randomised design. Comparison of sperm parameters was performed by t-test and fertility by Fisher's exact test. No difference (P &gt; 0.05) was observed in total motility (%, CG = 57.9 ± 6.5 v. TG = 60.2 ± 6.7), progressive motility (%, CG = 26.9 ± 4.8 v. TG = 28.5 ± 4.8), percentage of rapid sperm (%, CG = 43.5 ± 8.8 v. TG = 45.7 ± 7.6), membrane integrity (%, CG = 20.1 ± 5.1 v. TG = 20.3 ± 6.3), and fertility (CG = 60% v. TG = 70%) between the groups. The results of this study showed that the use of cholesterol-loaded cyclodextrin did not affect sperm kinetic parameters and fertility in stallion with good quality in post-thaw semen. Further studies must be performed with stallions sensitive to freeze-thawing process.

69 REACTIVE OXYGEN SPECIES EVALUATION OF DONKEY FROZEN SEMEN ADDED TO HOMOLOGOUS SEMINAL PLASMA ON POST-THAW

For many years the pregnancy rate of donkey frozen semen presented lower results in donkey jennies; however, a recent study showed an increase in pregnancy rates using frozen semen added to seminal plasma on post-thaw. A hypothesis for this result is the higher uterine inflammation response after breeding when using seminal plasma. The same studies demonstrated higher uterine inflammation in the presence of higher reactive oxygen species concentration. The aim of the present study was to evaluate the content of reactive oxygen species in donkey frozen semen added to homologous seminal plasma on post-thaw. Five ejaculates from each 3 donkeys were used. Semen was diluted (1 : 1) with a skim milk-based extender (Botu-SemenTM, Botupharma, Brazil). The semen was frozen with Botu-CryoTM extender (Botupharma, Brazil) in an isothermal box in straws containing 100 × 106 of total sperm. The samples were thawed at 46°C for 20 s. After this, the straws of each donkey were divided in 2 group: control group (CG), in which the semen was incubated at 37°C for 5 min, and plasma seminal group (PG), in which the semen was incubated at 37°C for 5 min with 70% of homologous seminal plasma. Sperm kinetic parameters were evaluated by computer-assisted semen analysis, and the plasma membrane integrity (propidium iodide and fluorescein isothiocyanate -PSA) and reactive oxygen species (5–6-carboxi-2,7-diclorodihidrofluoresceindiacetate) were evaluated by flow cytometer. Comparison of sperm parameters was performed by t-test. Total motility (%, CG = 75.4 ± 8.2a v. PG = 57.5 ± 16.4b), progressive motility (%, CG = 42.0 ± 8.7a v. PG = 33.3 ± 13.2b), progressive angular velocity (μm/s, CG = 95.8 ± 10.8a v. PG = 88.9 ± 10.9b), and percentage of rapid sperm (%, CG = 58.4 ± 12.5a v. PG = 41.0 ± 17.3b) were higher in CG compare with PG. No difference (P &lt; 0.05) was observed in membrane integrity (%, CG = 20.7 ± 7.4 v. PG = 20.6 ± 7.8); however, reactive oxygen species (%, CG = 12.3 ± 10.6a v. PG = 81.8 ± 32.5b) were higher in PG. The results of this study showed that the addition of homologous seminal plasma on post-thaw decreases the sperm kinetic parameters and viability of donkey frozen semen but increases reactive oxygen species, and this may cause higher uterine inflammation response in donkey jennies and increase their fertility.

65 ARE “BAD FREEZER” STALLIONS ALSO “BAD COOLER” STALLIONS?

Several factors can interfere with sperm cryopreservation resistance, especially the genetic factors and those related to the plasma membrane composition of the sperm and seminal plasma. However, it is still unclear if the same factors that confer freezing resistance will perform the same role during the cooling process. Thus, the aim of this study was to determine the relation between the resistance to freezing and cooling processes in stallions. Two ejaculates from each of 75 stallions were used. All animals showed good quality of fresh semen (total motility higher than 60% and plasma membrane integrity higher than 50%). After collection, the semen was diluted 1 : 1 with commercial skim milk-based extender (Botu-SemenTM, Botupharma, Brazil) and then a part was designed to cooling and the another to freezing. The cooled semen was divided into 2 groups: Group PS, in which the semen was diluted with Botu-SemenTM at a concentration of 50 × 106 sperm mL–1, and Group SPS, which was subjected to a centrifugation at 600 × g for 10 min and resuspended with Botu-SemenTM at 50 × 106 sperm mL–1. Semen samples from both groups were placed in the same cooling passive system for a period of 24 h/5°C. To accomplish the freezing process, the semen sample was subjected to centrifugation at 600 × g for 10 min. The supernatant was discarded, and the pellet was re-suspended in a Botu-CrioTM. The straws were frozen according to the manufacture. The sperm parameters from fresh semen, cooled semen for 24 h with and without seminal plasma, and frozen semen were evaluated for kinetics by computer-assisted semen analysis and for plasma membrane integrity (IMP%) by epi-fluorescence microscopy. The animals were classified in relation to their resistance to cooling and freezing processes as follow: “bad coolers” – reduction in sperm total motility and in plasma membrane integrity higher than 35% after 24 h of cooling in samples with seminal plasma; “good coolers” – reduction in sperm total motility and in plasma membrane integrity lower than 35% after 24 h of cooling in samples with seminal plasma; “bad freezer” – sperm total motility lower than 40% and progressive motility lower than 20% in seminal sample after thawing; “good freezer” – sperm total motility higher than 60% and progressive motility higher than 30% in seminal sample after thawing. The comparison between the resistance to cooling and freezing processes was performed by Fisher's exact test. The level of significance was 5%. No difference (P &lt; 0.05) between the resistance to cooling and freezing processes was observed. The percentage of stallions “good freezer” and “good cooler” was 54%, “good freezer” and “bad cooler” was 22.6%, “bad freezer” and “good cooler” was 12%, and “bad freezer” and “bad cooler” was 10.6%. Within stallions classified as “good freezer” and “bad cooler,” 52.9% also were “good cooler” when the seminal plasma was removed before the cooling process, and 47.1% remained as “bad cooler.” The result of this study demonstrates that there is a strong relation between the resistance to cooling and freezing processes in stallions. In stallions categorized as “bad cooler,” the seminal plasma presents a major influence on the quality and longevity of cooled semen.

Ultrastructure evaluation of goat spermatozoa after freezing in a skim milk-based extender with Trolox supplementation

The aim of this work was to evaluate the in vitro effect of adding Trolox in freezing extender for goat semen. Ejaculates from five bucks were evaluated, and when approved, the samples were pooled, diluted according to experimental groups [Trolox 0 (control), 30, 60 and 120nmolml(-1) ] and frozen in an automated system. Thawed samples (37°C/30s) were evaluated for plasma membrane (PMi) and acrosome integrity (Aci), mitochondrial membrane potential (MMP) and sperm kinematics by CASA system. Spermatozoa ultrastructure was evaluated in fresh and post-thawed semen. No significant difference (P>0.05) was observed among control and Trolox groups in the analyses of PMi, Aci, MMP and CASA in goat spermatozoa after thawing. Samples of 60 and 120nmolml(-1) Trolox groups had a higher percentage of cells that had intact plasma membranes in spermatozoa head than in the other groups, although they did not differ (P>0.05) before being frozen. A higher percentage (P<0.05) of spermatozoa with intact mitochondria was observed in fresh semen, control and Trolox 60nmolml(-1) groups than in the other groups. Addition of Trolox to skim milk extender at 60nmolml(-1) ultrastructurally preserves the plasma membrane and mitochondrial sheath integrity in goat spermatozoa after cryopreservation.

Influence of cholesterol-loaded cyclodextrin incorporation on the acrosome reaction of cooled equine spermatozoa

Sperm capacitation with subsequent acrosome reaction is an indispensable event for oocyte fertilization. To trigger such events, cholesterol efflux from the membrane is necessary. Although cholesterol-loaded cyclodextrin (CLC) incorporation to the membrane increases sperm resistance to cooling, it might also delay or inhibit sperm capacitation and acrosome reaction. The goal of this study was to assess CLC addition on the acrosome reaction of cooled equine spermatozoa after challenge with calcium ionophore. For this purpose, two ejaculates from five stallions were collected. Each ejaculate was diluted to 50 x 106 sperm/mL with skim milk-based extender and divided into two 5-mL aliquots. For each ejaculate, one aliquot was not treated

Comparison of sperm parameters and fertility between sperm cryopreserved from ejaculated semen and from the epididymis of subfertile stallions

Despite the benefits provided by seminal plasma during natural cover, its function is questionable when a biotechnology such artificial insemination is used. Studies employing spermatozoa from the cauda epididymis with or without the addition of seminal plasma showed no difference in conception rates. Furthermore, studies have reported deleterious effects of secretions from accessory glands on sperm viability and conception rate (Aurich et al., Theriogenology, 1996; 46, 791-797), especially in stallions with poor semen quality. Thus the aims of this study were: 1) compare the sperm kinetic parameters, viability and fertility of sperm recovered from the ejaculate (G1) and from the cauda epididymis (G2) of subfertile stallions (pregnancy rate<35%). For G1, one ejaculate from each of eight subfertile stallions was collected and then subjected to cryopreservation using the extender BotuCrioTM. One week after the last semen collection, the stallions underwent bilateral orchiectomy. Sperm from the cauda epididymis were harvested immediately after castration by retrograde flushing the caudal portion (G2) of the epididymis using a skim milk-based extender (BotuSemenTM). The recovered sperm were then cryopreserved using BotuCrioTM extender. The sperm samples were analyzed for kinetic parameters by computer-assisted analyses (CASAHTM-IVOS) and plasma membrane integrity (Propidium iodide, Sigma; Hoechst 33345, Sigma), acrosome integrity (FITC-PSA, Sigma; Hoechst 33345, Sigma) and degree of translocation of phosphatidylserine (Annexin-V–APC, Pharmingen; Hoechst 33345, Sigma) by flow cytometry (BD-LSRFortessa ). For analysis, straws were thawed in a 46 C water bath for 20 sec. Artificial insemination was performed in the tip of the uterine horn ipsilateral to

Improvement of cooled equine semen by addition of carnitines

L-carnitine (LC) and acetyl-L-carnitine (AC) modulate several sperm metabolic functions, such as fatty acid oxidation, acetyl-CoA/free CoA ratio and utilization of pyruvate and lactate as energy substrates. LC has a powerful antioxidant effect by reducing the availability of lipids to peroxidation and increasing activity of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase. Moreover, the presence of carnitines ensures the operation of oxidative pathways by reducing acetylCoA levels and provides acetyl groups for sperm motility. The aim of this study was to evaluate the effect of these substances on cooled spermviability. Two ejaculates from 6 stallions were diluted with skim milk-based extender (Botu-SemenTM) to a final concentration of 50x106 spermatozoa/mL and then divided into 4 groups: Control (no LC/AC), LC (0.1mM/mL of LC), AC (0.1mM/mL of AC) and LC/ AC (0.1mM/mL of LC+0.1mM/mL of AC). All groups had the osmolarity adjusted to 380 mOsm and pH at 6.8. After treatment, samples were placed at 5 C for 24 and 48 hr. Sperm motility was determined by computer-assisted semen analysis (Hamilton-ThorneTM) for total motility

The effect of dietary n-3 polyunsaturated fatty acids supplementation of rams on semen quality and subsequent quality of liquid stored semen

The objective of this study was to examine the effect of dietary n-3 polyunsaturated fatty acid (PUFA) supplementation of rams on semen quality and subsequent sperm function of liquid stored semen. Mature rams of proven fertility were individually housed and were blocked according to breed, body weight, and body condition score and randomly allocated within block to one of two dietary treatments (N = 7 per treatment). Rams were offered a base diet of hay and concentrate, with the concentrate enriched with either: (1) saturated palmitic acid (CON) or (2) high n-3 PUFA fish oil (FO) supplements. Both lipid supplements were added at 2% (wt/wt) of the total diet as fed and both were partially rumen-protected. The animals were fed their respective diets for a total of 9 weeks and blood samples were collected on weeks 0 (pre-experimental), 4, and 9, relative to initial allocation of diet (week 0), for measurement of plasma concentration of fatty acids, metabolites, insulin like growth factor 1 (IGF-1) and insulin. Semen was collected from each ram (on 1 day in each week) in weeks 4, 5, 7, 8, and 9, and each ejaculate was assessed for volume, wave motion, and concentration of sperm, after which it was diluted in a skim milk-based extender and stored at 4 °C. A second ejaculate was collected on weeks 4, 7, and 9, centrifuged, and the sperm frozen for subsequent lipid analysis. A sample of semen from each ram was assessed at 24, 48, and 72 hours after collection for sperm progressive linear motion, ability to penetrate artificial mucus, and the ability to resist lipid peroxidation (at 24 and 48 hours only) using the thiobarbituric acid reactive substances assay. There was no effect of diet on plasma insulin concentrations or on any of the metabolites measured, however, there was a diet by week interaction for plasma IGF-1 concentration (P < 0.05). This was manifested as the FO supplemented rams having higher IGF-1 concentrations on week 9 compared with the control treatment (P < 0.05), but not at the earlier sampling dates. Compared with the pre-experimental values, supplementation with FO increased plasma concentrations of total n-3 PUFAs by 3.1-fold and decreased n-6 PUFA concentrations by 1.84-fold. Consequently, the ratio of n-6 to n-3 PUFA was decreased in the FO-supplemented rams (P < 0.001). Dietary supplementation with FO increased the concentration of eicosapentaenoic acid in sperm from week 4 to 9 by 2.7-fold (P < 0.05) leading to a 1.5-fold increase in total n-3 PUFA in the same period. Ejaculates collected from rams supplemented with FO yielded a higher semen concentration (P < 0.05), however, there was no difference between diets on any of the other semen quality parameters including semen volume, wave motion, progressive linear motion, ability to penetrate artificial mucus, or ability to resist lipid peroxidation. In conclusion, dietary supplementation of rams with n-3 PUFA successfully increased the n-3 PUFA content of plasma and sperm but has limited effects on the quality of liquid stored semen.

247 COMPARISON OF THE DNA FRAGMENTATION INDEX BETWEEN CRYOPRESERVED EJACULATED SPERM AND EPIDIDYMAL SPERM IN STALLIONS

The development of a reliable technique for freezing epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The semen analysis method with the best ability to predict fertility is an examination of the sperm chromatin structure. This test evaluates the susceptibility of spermatozoa DNA to denaturation. The ability of spermatozoal DNA to maintain an intact double-stranded configuration is determined by exposure to an acid environment. The aim of this study was to compare the DNA fragmentation index of sperm obtained from ejaculate (G1) and sperm from the cauda epididymis (G2). For G1, two ejaculates from each of seven stallions were collected and then subjected to cryopreservation using BotuCrioTM extender. One week after the last semen collection, the stallions underwent bilateral orchiectomy. Sperm from the cauda epididymis was harvested immediately after castration (G2) by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender (BotuSemenTM). The recovered sperm was then cryopreserved using BotuCrioTM extender. The sperm motility parameters were analysed by computer-assisted sperm analysis (HTM IVOS 12, Hamilton Thorne Inc., Beverly, MA, USA), and the DNA fragmentation index was estimated using acridine orange test epifluorescence microscopy. The samples were evaluated immediately (0 h) and 8 h after thawing. The total motility, progressive motility, and percentage of rapid cells of the G1 v. G2 samples at 0 h were, respectively, 62.3 ± 12.9a v. 72.6 ± 8.4a, 31.6 ± 9.2a v. 35.3 ± 10.32a, and 49.3 ± 14.33a v. 59.7 ± 13.59a. At 8 h, the results were 26.0 ± 21.6b v. 54.7 ± 12.2a, 6.1 ± 6.4b v. 17.4 ± 8.54a, and 13.7 ± 14.85b v. 37.6 ± 14.15a. Evaluation of the DNA fragmentation in the G1 and G2 samples yielded 6.7 ± 1.41a v. 5.7 ± 1.60a at 0 h and 8.3 ± 1.78b v. 7.2 ± 1.19b at 8 h for percentage of DNA fragmentation after thawing. At 0 h, no differences in the sperm parameters were observed between groups, but statistical differences were observed in the sperm motility parameters between the treatment groups after 8 h. For the DNA fragmentation index, no difference was found at 0 and 8 h between the groups. However, after thawing, a higher percentage of DNA fragmentation was observed in the ejaculated sperm (8 h) as compared with the epididymal sperm (0 h). On the basis of these results, we can conclude that frozen–thawed cauda epididymal sperm had similar or higher motion parameters than ejaculated sperm after thawing. In addition, incubating the sperm at 20°C for 8 h after thawing resulted in higher motion parameters and less DNA fragmentation of the epididymal sperm. This finding suggests that epididymal sperm are more resistant to the cold shock caused by cryopreservation. FAPESP for financial support and Botupharma for donation of BotuSemenTM and BotuCrioTM extender.

22 EFFECT OF CHOLESTEROL ADDITION TO EQUINE SPERM MEMBRANE ON FERTILITY

Artificial insemination with cooled-shipped semen has been widely used in horse breeding. However, some stallions, referred to as poor coolers, present abrupt fertility decrease when their semen is processed, cooled, and transported. Cholesterol incorporation into sperm membranes improves the quality of cryopreserved semen by increasing the sperm membrane stability and fluidity at low temperatures. Despite the beneficial effect of cholesterol addition on sperm quality, studies demonstrate that the presence of large amounts of cholesterol in the plasma membrane interferes with the physiological process of sperm capacitation and is detrimental to frozen equine sperm fertility. The aim of this study was to assess the fertility of cooled semen from good-cooler and poor-cooler stallions after adding cholesterol to sperm membranes. Two stallions were used and classified as good cooler (n = 1) and poor cooler (n = 1) based on the ability to maintain sperm progressive motility after 24 h of cooling at 5°C. For classification of the stallions, the fertility history was also taken into account through the results of pregnancy per cycle using inseminations with cooled semen (&lt;50% for poor cooler and &gt;70% for good cooler). Ejaculates of these stallions were subjected to 2 treatments: control (CON) and cholesterol (CLC). In the CON group, the semen was extended to 30 × 106 sperms mL–1 with skim milk-based extender (BotuSemen™). In the CLC group, the semen was extended as in the CON group plus 0.25 µL/1 × 106 sperms of 6.1 mM cholesterol-loaded cyclodextrin was added. Afterwards, both treatments were cooled at 5°C for 24 h. To test the fertility of poor-cooler and good-cooler stallions, 2 cycles from 25 mares and 2 cycles from 10 mares were respectively used. For both stallions, randomly for each mare, the inseminations were performed by alternating both treatments. If the mare was first inseminated with the CLC treatment, in the next cycle the CON treatment was used and vice versa. After 24 h of ovulation induction, the inseminations were done in the uterine body with 1 × 109 viable cells. Statistical analyses were performed using the Fisher exact test and significance was set at P &lt; 0.05. For the poor cooler, the CON treatment presented 44% pregnancy/cycle compared to 76% for the CLC treatment (11/25a v. 19/25b). For the good cooler, both treatments presented 80% (8/10) pregnancy/cycle. The results suggest that the fertility capability of stallions is not affected by incorporation of cholesterol-loaded cyclodextrin to the sperm plasma membrane. Additionally, the utilization of cholesterol-loaded cyclodextrin may be an option to enable the utilization of cooled-shipped semen from poor cooler stallions for AI programs.