Paradoxical Effect of Quercetin Antioxidant on Goat Sperm Parameters After Cryopreservation.

BACKGROUND: Some antioxidants have been used in semen extenders to reduce adverse effects caused by excessive reactive oxygen species (ROS) production. The study was carried out to assess the effect of quercetin (QC) antioxidant therapy on goat semen submitted to cryopreservation. OBJECTIVE: To evaluate the effect of quercetin incorporation in different phases of the cryopreservation process of goat spermatozoa. MATERIALS AND METHODS: Five ejaculates from each of four goats (n= 20) were collected and split into four groups: Control (G1), without QC; G2, 15 µM of QC added to semen before centrifugation; G3, 15 µM QC added to semen after centrifugation; G4, 15 µM QC added to semen before centrifugation and 15 µM of QC added to semen after centrifugation (total of 30 µM of QC); and cryopreserved. All semen samples were evaluated after thawing for sperm kinetics, plasma membrane integrity, and ROS levels. RESULTS: Although lower concentrations of ROS were associated with groups that received antioxidant supplementation (P=0.0213), linear and dose dependent (P<0.05) reductions of the total and progressive sperm motility, velocity and percentage of fast cells were related to the QC groups. Likewise, plasma membrane integrity was better preserved (P=0.0154) in the control group (35.5%) than in groups that received QC (G2=32.6%, G3=32.4% and G4=26.7%). CONCLUSION: Although quercetin was efficient at reducing the oxidative stress related to sperm cryopreservation, it exerted a deleterious dose-dependent effect on the kinetics and integrity of the frozen goat semen, contradicating its use in the tested concentrations.

Pentoxifylline effects on capacitation and fertility of stallion epididymal sperm.

The aims of this study were to determinate whether pentoxifylline (PTX) increases the motion parameters of fresh and frozen-thawed equine epididymal spermatozoa, to evaluate the tyrosine phosphorylation of frozen-thawed epididymal sperm in the presence of PTX and to determine whether the PTX-treatment of stallion epididymal sperm prior to freezing improves the fertility response of mares to a reduced number of spermatozoa per insemination dose. Fifty epididymis were flushed with a skim milk based extender with or without PTX. The pre-treatment with PTX enhanced the sperm motility after being harvested (P<0.05); however the freeze-thaw process did not alter the sperm kinematics between control and treated samples (P>0.05). Plasma membrane integrity did not differ between control and PTX group after recovery and after thawing (P>0.05), as observed in tyrosine phosphorylation, which the PTX treatment did not alter the percentage of tail-associated immunofluorescence of cryopreserved epididymal sperm (P>0.05). For the fertility trial, different insemination groups were tested: 800×106 epididymal sperm (C800); 100×106 epididymal sperm (C100); 100×106 epididymal sperm recovered in an extender containing PTX (PTX100). The conception rates for C800; C100 and PTX100 were 68.7% (11/16); 31.5% (5/16) and 50% (8/16), respectively. The conception rate did not differ among groups (P>0.05), however, a low number of animals was used in this study. A trend toward significance (P=0.07) was observed between C800 and C100 groups. In conclusion, PTX has no deleterious effect on sperm motility, viability and capacitation of cryopreserved stallion epididymal sperm. The conventional artificial insemination with 100×106 sperm recovered with PTX ensures acceptable conception rates and maximize the limited number of doses of cryopreserved stallion epididymal sperm.

Seminal plasma arising from the whole boar sperm-rich fraction increases the stability of sperm membrane after thawing.

Boar spermatozoa arising from the sperm-rich ejaculate fraction are reported to have a more stable plasma membrane and are more resistant to cold shock and premature acrosome reaction than spermatozoa from the whole ejaculate. Furthermore, seminal plasma (SP) can increase the cryotolerance of boar spermatozoa, and in other domestic species, it has the ability to reverse cryopreservation damage. This study aimed to evaluate the effects of boar SP arising from the whole sperm-rich ejaculate fraction (SP-SRF) on the integrity, stability, and peroxidation of sperm membranes after thawing. Each ejaculate ( = 24) was divided among 4 treatments: control (CT), centrifuged and suspended in autologous SP-SRF (CS), centrifuged with withdrawn SP-SRF (CW), and post-thawed SP arising from the whole sperm-rich fraction addition to CW (CWSP). After thawing, all treatments were incubated for 5, 60, and 120 min and were analyzed for membrane integrity, fluidity, and peroxidation by flow cytometer. The absence of SP-SRF increased the lipid disorder ( < 0.05) but had no effect on lipid peroxidation ( > 0.05) or membrane integrity ( > 0.05). However, the increase in lipid disorder by withdrawal of SP-SRF was reversed by SP-SRF addition ( < 0.05) to the post-thawing medium, whereas plasma and acrosomal membrane integrity ( > 0.05) and lipid peroxidation ( > 0.05) were unchanged. In conclusion, despite the centrifugation effects, the addition of SP arising from the whole sperm-rich fraction to post-thawed boar semen decreased sperm lipid disorder without an influence of the sperm membrane integrity and peroxidation.

Sperm fertility and viability following 48h of refrigeration: evaluation of different extenders for the preservation of bull semen in liquid state.

Two experiments were conducted to compare the effectiveness of different extenders conventionally used for semen cryopreservation to maintain the viability and fertility of cooled bull semen. In Experiment 1, sperm samples obtained from 20 Nellore bulls were preserved at 5°C for 48h using two extenders containing 20% of egg yolk [Tris (TRIS-R) and Botu-Bov(®) (BB)] and another composed of 1% soy lecithin [Botu-Bov(®)-Lecithin (BB-L)] as substitutes for animal origin products. The samples were evaluated at 6, 24 and 48h for plasma and acrosomal membrane integrity, quantification of thiobarbituric acid reactive substances (ng of TBARS/10(8) cells) and sperm motility parameters by computer-assisted semen analysis (CASA). In Experiment 2, pregnancy rate (P/AI) of 973 fixed-time artificially inseminated Nellore cows were compared when cows were inseminated with conventionally cryopreserved semen in TRIS-egg yolk glycerol (TRIS-C Control, n=253) or semen cooled for 48h in TRIS-R (n=233), BB (n=247) or BB-L (n=240). Although none of the extenders used was effective on maintaining total progressive motility and cellular integrity throughout the 48-h of the refrigeration period (P<0.01), BB-L conferred greater protection against oxidative stress (P<0.05) than egg yolk-based medias. The P/AI for semen samples preserved in TRIS-C, TRIS-R, BB and BB-L were 39.92(a), 25.32(b), 26.32(b) and 33.33(ab), respectively. These results demonstrate that the three conventional extenders used for semen cryopreservation do not provide the protection required to maintain bull semen fertility under refrigeration for a 48-h period, resulting in reduced pregnancy rates. However, the use of lecithin-based medium instead of egg yolk results in greater protection against lipid peroxidation, producing P/AI results comparable to those obtained using frozen semen.

Use of cholesterol-loaded cyclodextrin: an alternative for bad cooler stallions.

During the cooling process, sperm may suffer irreversible damage that compromises the fertility rate. Incorporating cholesterol-loaded cyclodextrin (CLC) represents a strategy to increase sperm resistance at low temperatures; however, high levels of cholesterol in the cell membrane can interfere with sperm capacitation. The goals of this study were to determine the CLC concentration and cooling temperature that produce optimal kinetic parameters and viability of sperm from stallions identified as bad coolers (BCs) and good coolers (GCs), as well as the effect of adding CLC on the occurrence of the acrosome reaction (ACR) and on the fertility rate of cooled sperm. In experiment 1, each ejaculate was divided into four groups: Control and treated with 1 (CLC-1), 1.5 (CLC-1.5), or 2 mg (CLC-2) of CLC/120 × 10(6) sperm and cooled for 48 hours at 5 °C. In experiment 2, each ejaculate was divided into four groups: Control and CLC-1.5 cooled at 15 °C or 5 °C for 24 hours. For experiment 3, GC and BC stallions were used, and the ejaculates were divided into control and CLC-1.5 cooled at 5 °C for 48 hours. According to experiment, the sperm kinetics (SK) and plasma membrane integrity (PMI) were analyzed before and after 24 and 48 hours of cooling. In experiment 4, the ejaculates were divided into four groups: Control and CLC-1.5 maintained at room temperature or cooled at 5 °C for 24 hours. Each group was incubated with ionophore calcium at 37 °C for 3 hours. The incidence of ACR was analyzed before and after 1, 2, and 3 hours of incubation. For experiment 5, two cycles of 10 mares for a GC stallion and two cycles of 25 for a BC stallion were used. The inseminations were performed with control and CLC-1.5 groups cooled at 5 °C for 24 hours. According to results, all groups treated with CLC exhibited higher PMI compared with controls, and CLC-1.5 and CLC-2 exhibited the best SK results. The cooling temperature of 5 °C was superior to 15 °C when the sperm was treated with CLC. The GC and BC stallions benefited from the CLC-1.5 treatment, but the BCs were more evident, which presented greatly increased PMI and SK. There was a delay in capacitation of at least 3 hours for the fresh sperm and at least 1 hour for cooled sperm supplemented with CLC-1.5. After adding CLC-1.5, the fertility of BC stallion significantly increased, but that of the GC was not altered. Thus, incorporating CLC is an effective technique to cool equine semen, although it is indicated mainly for BC stallions.

New seminal plasma removal method for freezing stallion semen.

Seminal plasma removal, an indispensable step in equine semen cryopreservation, is usually done by centrifugation, but this might cause mechanical damage to sperm. A new method for seminal plasma removal from stallion semen, namely a filter composed of a synthetic hydrophilic membrane (Sperm Filter, BotuPharma, Botucatu, Sao Paulo, Brazil), was recently proposed. The objective of this study was to test the use of the Sperm Filter in the removal of seminal plasma before freezing stallion semen. Ejaculates from 31 stallions were divided into two groups and cryopreserved. In group 1 (G1), seminal plasma was removed with the Sperm Filter, and in group 2 (G2), seminal plasma was removed by centrifugation (600×g for 10 minutes). There were no differences (P < 0.05) between G1 and G2 in sperm kinetic parameters or plasma membrane integrity before or after cryopreservation. However, sperm recovery rate was higher (P < 0.05) for G1 versus G2 (mean ± SD, 89.4 ± 7.4% vs. 80.9 ± 5.5%). Therefore, the Sperm Filter was as efficient as centrifugation in removing seminal plasma from the stallion ejaculate. However, filtering was more practical and had significantly fewer sperm lost than the centrifugation technique.

Induction of double ovulation in mares using deslorelin acetate.

This study aimed to determine whether deslorelin acetate could induce double ovulation in mares. In Experiment 1, eight mares were treated with prostaglandin on Day 8 (D8) after ovulation, then treated with saline or with 100 µg of a controlled-release formulation of deslorelin acetate vehicle intramuscularly (IM) every 12h from D8 after ovulation until at least two follicles reached 33 mm. At this time, ovulation was induced with 2500 IU of hCG. Artificial insemination was performed 24h after induction, and embryos were collected on the eighth day after ovulation was first detected. In Experiment 2, 112 estrous cycles in 56 mares were studied. In this experiment, the deslorelin acetate protocol was initiated only in mares that achieved a follicle with a diameter of at least 25 mm and at least one second follicle with a diameter≥20mm was detected, at which time 100 µg deslorelin acetate or saline was administered IM every 12h. The other procedures were similar to those described in Experiment 1. The variables studied were analyzed using Student's t-test and Fisher's exact test. In Experiment 1, only two mares in deslorelin group having second follicles of 20-25 mm on responded with double ovulation. In the second experiment, 82% of treated mares responded with double ovulation, and the embryo recovery per estrous cycle was 1.12 and 0.57 in the group treated with deslorelin acetate and the control group, respectively (P<0.05). Deslorelin acetate is effective in inducing double ovulation in mares using the protocol proposed. On average, it allows for the recovery of one embryo by uterine flushing.

Thermoresistance sperm tests are not predictive of potential fertility for cryopreserved bull semen.

Different studies demonstrate positive correlations between seminal variables determined in the laboratory and subsequent fertility after artificial insemination. It is clear, however, that there is still a deficiency in predicting in vivo fertility results of semen samples. The present study intended to verify the efficiency of rapid and slow thermoresistance tests in predicting fertility of frozen semen of bulls. Sperm from 64 ejaculates of 39 Nelore bulls (Bos indicus), aged 2-10 years, were cryopreserved in 0.5 mL straws. Thawed straws containing 30 x 10(6) sperm were analyzed for seminal variables in the laboratory and used to inseminate 4920 cows to evaluate fertility in the field. The ejaculates were frozen in a Tris-based extender and samples were evaluated for total motility after rapid (46 degrees C/30 min) and slow (38 degrees C/5h) thermoresistance tests by conventional and computerized (CASA) methods. Sperm samples were grouped according to their ability to retain motility after thermoresistance testing: group 0 (0% motility), group 1 (1-20% total motility), group 2 (21-40% total motility) and group 3 (>40% total motility). Correlation and association between these groups and fertility diagnosed by rectal palpation at 90 days were verified. Chi-square test demonstrated no association between motility groups and fertility (P>0.25) and both rapid and slow thermoresistance tests had a lesser correlation to fertility (r=0.11 and 0.14, respectively). These results demonstrated that these tests are not reliable in predicting in vivo behavior of bull frozen semen and are not effective to estimate fertility.

Freezing of stallion epididymal sperm.

Inseminations with frozen-thawed epididymal sperm have resulted in low-pregnancy rates of mares. If fertility of epididymal sperm could be improved, it would help to preserve genetic material from stallions that have suffered severe injuries, been castrated or have died. The aim of the present study was to investigate the effect of different extenders and pre-freezing addition of capacitation media on freezability of epididymal sperm and on storage at 5 degrees C for 24h. In experiment 1, epididymal sperm samples were diluted and subsequently frozen with three different extenders: Botu-Crio, EDTA-Lactose and INRA-82. Motility analysis using computer assisted sperm analyzer (CASA) demonstrated better motility for sperm in Botu-Crio than in the other extenders; EDTA-Lactose yielded better motility than INRA-82 on most evaluated parameters. There was no difference in membrane integrity among the studied extenders. From 18 inseminated mares, 12 (66%) were pregnant 15 days after AI with frozen-thawed epididymal sperm showing that Botu-Crio was able to maintain the fertility potential. In experiment 2, the effect of incubation of epididymal sperm before freezing in three capacitation media (Fert Talp, Sperm Talp, Talp+Progesterone), seminal plasma, or control was tested. Based on post-thaw motility evaluation by CASA, samples incubated in Sperm Talp showed better motility values. There were no differences in plasma or acrosomal membranes or in mitochondrial potential among groups. We concluded that Botu-Crio was better than the other extenders in the ability to preserve epididymal sperm and that pre-freeze addition of Sperm Talp was also beneficial.

Ovulation synchronization following commercial application of ultrasound-guided follicle ablation during the estrous cycle in mares.

A regimen of progesterone plus estradiol (P&E) was used as a standard for ovarian synchronization to test the efficacy and evaluate the commercial application of ultrasound-guided follicle ablation as a non-steroidal alternative for ovulation synchronization in mares. Recipient mares at a private embryo transfer facility were at unknown stages of the estrous cycle at the start of the experiment on Day 1 when they were randomly assigned to an ablation group (n=18-21 mares) or to a P&E group (n=20-21 mares). In the ablation group, mares were lightly sedated and all follicles > or = 10 mm were removed by transvaginal ultrasound-guided follicle aspiration. In the P&E group, a combination of progesterone (150 mg) plus estradiol (10mg) prepared in safflower oil was given daily (im) for 10 d. Two doses of prostaglandin F(2alpha) (PGF, 10mg/dose, im) were given 12 h apart on Day 5 in the ablation group, or a single dose on Day 10 in the P&E group. Human chorionic gonadotropin (hCG, 2500 IU/mare, im) was given at a fixed time, 6 and 10 d after PGF treatment in the ablation and P&E groups, respectively, with the expectation of a follicle > or = 30 mm at the time of treatment. In both the ablation and P&E groups, transrectal ultrasonography was done at the start of the study (Day 1) and again on the day of hCG treatment and daily thereafter to determine the presence of a CL, measure diameter of the largest follicle and detect ovulation. The mean interval from the start of the study and from PGF treatment to ovulation was shorter (P<0.0001) in the ablation group (13.7 and 9.7 d, respectively) compared to the P&E group (22.3 and 13.2 d, respectively). Following fixed-day treatment with hCG after PGF treatment, the degree of ovulation synchronization was not different (P>0.05) between the ablation and P&E groups within a 2-d (56 and 70%) or 4-d (83% and 90%) period. Although ultrasound-guided follicle ablation may not be practical in all circumstances, it excluded the conventional 10-d regimen of progesterone and estradiol and was considered an efficacious and feasible, non-steroidal alternative for ovulation synchronization in mares during the estrous cycle.