Evaluation of used Purslane extracts in Tris extenders on cryopreserved goat sperm

This study aimed to evaluate the comparative effects of Purslane aqueous extract (PAE), Purslane methanolic extract (PME) and Purslane ethanolic extract (PEE on the quality of frozen-thawed goat spermatozoa. Collected semen with motility >75% and sperm concentration >1.0 × 10⁹ sperm/ml was pooled and divided into 10 equal aliquots and supplemented by basic extender containing 25, 50 or 100 μg/ml of Purslane aqueous extract (PAE_25μg/ml, PAE_50μg/ml, PAE_100μg/ml, respectively), basic extender containing 25, 50 or 100 μg/ml of Purslane methanolic extract (PME_25μg/ml, PME_50μg/ml, PME_100μg/ml, respectively), basic extender containing 25, 50 or 100 μg/ml of Purslane ethanolic extract (PEE_25μg/ml, PEE_50μg/ml, PEE_100μg/ml, respectively). Control diluent contained no additives. For the determination of sperm quality, frozen straws were thawed and then the sperm characteristics were assessed. Results indicated that higher (P < 0.05) percentages of total motility, viability, mitochondrial activity and lower percentages of malondialdehyde (MDA) for PAE_50μg/ml, PME_50μg/ml and PEE_50μg/ml than those of the control. In addition, PME_50μg/ml resulted in the highest) P < 0.05) total motility and the lowest (P < 0.05) MDA levels compared to other treatments. Compared to the control group, PME_50μg/ml resulted in higher integrity (P < 0.05) of plasma membranes and in lower amounts of apoptotic and dead spermatozoa. PME_50μg/ml and PAE_50μg/ml showed higher (P < 0.05) percentages of progressive motility, DNA integrity and live post-thawed spermatozoa than those of the control. No significant differences in the motility, viability, mitochondrial activity and number of live sperms were observed between PME_50μg/ml and PAE_50μg/ml treatments. In conclusion, the results of this study indicated that 50 μg/ml purslane extracts could be used for the cryopreservation. However, the results of methanolic extract was more beneficial compared to other extracts.     

In vitro fertility evaluation of cryopreserved ram semen and its correlation with relative in vivo fertility

The present study was designed to evaluate zona-free hamster ova assay conditions for cryopreserved ram semen and to investigate the correlation between ability to penetrate zona-free hamster ova and in vivo fertility. In vivo fertility was estimated for cryopreserved semen from 5 Merino rams using heterospermic insemination. Equal numbers of postthaw motile spermatozoa from a Merino ejaculate and pooled Suffolk ejaculates were mixed prior to insemination. Each Merino ejaculate was paired with the same pool of cryopreserved Suffolk semen. Relative in vivo fertility for each Merino ram was calculated as the proportion of offspring that were sired by the Merino (range 42 to 100%). These ejaculates also differed in their ability to penetrate zona-free-hamster ova (3.6 to 9.0 penetrated spermatozoa per ovum). Differences in penetration rate were correlated with in vivo fertility (P < 0.002, R2 = 0.69). Results of these studies suggest that the zona-free hamster ova bioassay may be a useful test in the assessment of cryopreserved ram sperm fertility.     

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.     

Effect of sperm numbers and time of insemination relative to ovulation on sow fertility

We examined the effect of inseminating mixed parity sows (n = 231) once with fewer sperm at different times relative to ovulation. Lactation length was 19 days and sows received an IM injection of 600 IU equine chorionic gonadotrophin (eCG) 12 h before weaning. At 80 h after eCG injection, sows received an IM injection of 5 mg porcine luteinizing hormone (pLH). Predicted time of ovulation (PTO) was 38 h after pLH injection. Sows were assigned by parity to receive a single transcervical artificial insemination (AI) at either 6 or 24 h before PTO with semen doses containing either 2.5 or 1.25 × 10⁹ sperm. A positive control group of sows (n = 49) was subject to conventional AI 24 and 6 h before PTO. Detection of estrus was performed in the presence of a boar and only sows exhibiting estrous behavior at the assigned time of AI were included in the study. Farrowing rate for sows receiving 2.5 × 10⁹ sperm at 6 h before PTO was greater than that for sows receiving 1.25 × 10⁹ sperm at 24 h before PTO (85% versus 61%, P < 0.05). All other groups were intermediate. There was no effect of time of AI or sperm numbers on subsequent litter size. These data indicate that single insemination of fewer sperm may compromise sow fertility, even when performed transcervically, if not appropriately timed relative to ovulation.     

Morphological abnormalities in zebrafish cryopreserved sperm

The aim of the present study was to evaluate the effect of cryopreservation on the morphology of zebrafish sperm (Danio rerio). Sperm from 30 males were collected and divided in two treatments: fresh and cryopreserved semen. The following were measured sperm morphology, motility and membrane integrity. Cryopreservation reduced motility, the number of normal cells and the membrane integrity, as well as increased the percentage of sperm abnormalities. The most frequent types of morphological changes found in cryopreserved semen were macrocephaly, loose head, degenerated head, proximal gout, curled tail and short tail. This study opens the way for further investigations on morphological changes and for a new classification of these changes in fish semen due to cryopreservation.     

DNA integrity of Polyodon spathula cryopreserved sperm

Comet assay was used to detect DNA integrity of paddlefish (Polyodon spathula) sperm following cryopreservation. At the same time, sperm velocities prior to freezing and post‐thawing were also assessed by the computer‐assisted sperm analysis (CASA) system. Significant differences (P < 0.05) were detected in the degree of DNA damage in cryopreserved sperm using different extenders. According to osmolality of the extenders, DNA damages of Sb (20 mm Tris, 75 mm sucrose, 0.5 mm KCl, pH 8.5) sperm was the least, which showed that the percentage of tail DNA of Sb (17.87–35.28%) was lower than those of Sa (20 mm Tris, 50 mm sucrose, 0.5 mm KCl, pH 8.5) and Sc (20 mm Tris, 100 mm sucrose, 0.5 mm KCl, pH 8.5). Moreover, A and B class sperm cells provided most of the Sb sperm (>50%). However, in light of the concentration of methanol, DNA damages of M8 (8% methanol concentration) sperm were the least, including a lower percentage of the tail DNA (21.56–30.86%), and C and D class sperm cells (<30%), regardless of the osmolality of the extenders. In conclusion, when the dilution was 20 mm Tris, 75 mm sucrose, 0.5 mm KCl, pH 8.5 and the concentration of methanol was 8%, the extenders were the best for cryopreservation of paddlefish sperm. In addition, the results indicated that the extent of damage to sperm motility caused by freeze‐thawing (VCL, VSL) was correlated with DNA breakage (|r| > 0.8). This implied that cryopreservation could damage sperm DNA of paddlefish and affect the sperm velocities when the osmolality and the concentrations of the cryoprotectants of the extender were inappropriate.     

Apoptosis in fresh and cryopreserved buffalo sperm

The objectives of present study were (a) validation of annexin V/PI assay for estimation of sperm apoptosis in buffalo (Experiment 1) and (b) determining the effect of stages of cryopreservation on sperm apoptosis and its correlation with sperm motility and plasma membrane integrity (Experiment 2). In Experiment 1, different levels of apoptosis were artificially induced in buffalo semen (100 × 10⁶ sperm/aliquot) through graded doses of camptothecin (5, 10 and 20 μM/aliquot). Higher concentrations of camptothecin (10 and 20 μM) successfully (P < 0.05) induced apoptosis as compared to the lower (5 μM) dose and/or control. In Experiment 2, semen samples (n = 9, three pooled semen samples from each of the three buffalo bulls separately) were cryopreserved using vapor freezing. The mean percentage of apoptotic, necrotic and viable sperm did not differ between fresh and before freezing stages. However, freezing and thawing increased (P < 0.05) the percentage of apoptotic sperm (25.4 ± 0.6 vs. 36.5 ± 1.9) while decreased (P < 0.05) the necrotic (35.1 ± 1.2 vs. 29.7 ± 0.7) and viable sperm (37.2 ± 1.3 vs. 32.8 ± 1.9, (P < 0.07). Likewise, the mean percent motility and plasma membrane integrity decreased (P < 0.05) (64 ± 2.1 vs. 49.4 ± 1.3) and (79.6 ± 0.5 vs. 38.7 ± 0.3) respectively, at post thaw compared to other stages. Coefficient of correlation, combined at all stages for each variable revealed that sperm apoptosis was inversely correlated with sperm motility and plasma membrane integrity. It is concluded that (a) the annexin V/PI assay can be used as a tool to determine the buffalo semen apoptosis and (b) freezing and thawing induces apoptosis in buffalo sperm.     

Effect of preincubation of cryopreserved porcine epididymal sperm

Preincubation of spermatozoa is important for capacitation and successful fertilization in vitro. The effects of preincubation time on frozen-thawed boar epididymal spermatozoa as measured by sperm motility, acrosomal integrity and fertilization ability in vitro were examined. Epididymal spermatozoa were collected from three Large White boars and frozen. The thawed spermatozoa were preincubated for 0, 15, 30, 60 and 120 min. Their motility was evaluated by a sperm motility analyzer and then the sperm motility indexes (SMIs) were calculated. The status of their acrosomal integrity was evaluated by triple-staining. Then, their fertilization ability was examined by in vitro fertilization (IVF) using porcine oocytes matured in vitro. SMIs of spermatozoa and the incidences of acrosome-intact live spermatozoa from the three boars were high (21-39 for SMI and 50-61% for acrosome-intact live spermatozoa) just after thawing, but both decreased as the duration of preincubation was prolonged (2-10 and 23-40%, respectively). The incidences of sperm penetration were high (61-89% of inseminated oocytes) when the sperm were preincubated for 0-60 min. However, sperm penetration decreased as the preincubation period was prolonged to 120 min. The degree of this decrease differed depending upon the boar from which the spermatozoa were obtained (10-72%). When the two parameters, sperm motility and acrosomal integrity, were analyzed statistically, the latter parameter rather than the former one showed a significant effect on penetration ability in vitro after each duration of preincubation. These results suggest that preincubation of frozen-thawed boar epididymal spermatozoa is not required for IVF and also that the maintenance of acrosomal integrity in unreacted status, rather than the maintenance of sperm motility, is important for fertilization ability after thawing and during preincubation of boar epididymal spermatozoa.     

Subtle membrane changes in cryopreserved bull semen in relation with sperm viability,chromatin structure,and field fertility

This study investigated the use of annexin-V/PI assay to assess sub lethal changes in bull spermatozoa post-thawing, and to further relate these changes to results obtained by fluorometric assessment of sperm viability and sperm chromatin structure assay (SCSA), as well as field fertility (as 56-day non-return rates, 56-day NRR) after AI. Frozen-thawed semen samples were obtained from 18 Swedish Red and White bulls (one to three semen batches/bull) and fertility data was based on 6900 inseminations. The annexin-V/PI assay revealed that post-thaw semen samples contained on average 41.8+/-7.5% annexin-V-positive cells. Most of the annexin-V-positive cells were dying cells, i.e. also PI-positive. The incidence of annexin-V-positive cells was negatively related (r=-0.59, P<0.01) to the percentage of viable cells, as detected by fluorometry. The incidence of annexin-V-positive spermatozoa significantly correlated to the SCSA variable xalphat (r=0.53, P<0.05). The incidence of annexin-V-negative, dead cells was the only annexin-V/PI assay variable that correlated significantly with fertility both at batch (r=-0.40, P<0.05), and bull (r=-0.56, P<0.05) levels. Among sperm viability variables, subjectively assessed sperm motility (r=0.52-0.59, P<0.01), CASA-assessed sperm motility (r=0.43-0.61, P<0.05), and the incidence of live spermatozoa, expressed as total numbers (r=0.39-0.54, P<0.05), or percentage values (r=0.68-0.68, P<0.01), correlated significantly with field fertility both at batch, and bull levels. Among the SCSA variables, only the COMP alphat correlated significantly (r=0.33-0.51, P<0.05) with fertility results. The results indicate a certain proportion of bull spermatozoa express PS on their surface after thawing, e.g. they have altered membrane function, and that the incidence of such cells is inversely correlated to sperm viability, and positively correlated to abnormal sperm chromatin condensation since they eventually undergo necrosis.     

In vitro fertilization with cryopreserved inbred mouse sperm

Sperm from C57BL/6J, DBA/2J, BALB/cJ, 129S3/SvImJ, and FVB/NJ inbred mice were cryopreserved in 3% skim milk/18% raffinose cryoprotectant solution. The post-thaw sperm from all strains were evaluated for their viability and fertility by comparing them against B6D2F1 sperm used as a control. The protocol used for freezing mouse sperm was effective in different strains, because the motility was decreased by 50% after cryopreservation similar to other mammalian sperm. However, the progressive motility and the fertility of each inbred strain were affected differently. The C57BL/6J, BALB/cJ, and 129S3/SvImJ strains were the most affected; their fertility (two-cell cleavage) decreased from 70%, 34%, and 84% when using freshly collected sperm to 6%, 12%, and 6% when using frozen/thawed sperm, respectively. Live newborns derived from frozen/thawed sperm were obtained from all strains in the study. These results corroborate the genetic variation among strains with regard to fertility and susceptibility to cryopreservation.     

Live rhesus offspring by artificial insemination using fresh sperm and cryopreserved sperm

Artificial insemination (AI) and the cryopreservation of sperm with full reproductive capabilities are vital in the armamentarium of infertility clinics and reproductive laboratories. Notwithstanding the fantastic successes with AI and sperm cryopreservation in numerous species, including humans and cattle, these assisted reproductive technologies are less well developed in other species of importance for biomedical research, such as genetically modified mice and nonhuman primates. To that end, AI at high efficiency in the rhesus macaque (Macaca mullata) and the successful cryopreservation of rhesus sperm is presented here, as are the complexities of this primate model due to differences in reproductive tract anatomy and gamete physiology. Cryopreservation had no effect on the ability of sperm to fertilize oocytes in vitro or in vivo. Post-thaw progressive motility was not affected by cryopreservation; however, acrosome integrity was lower for cryopreserved (74.1%) than for fresh sperm (92.7%). Fertilization rates did not differ when fresh (58.1%; n = 32/55) or cryopreserved sperm (63.8%; n = 23/36) were used for in vitro fertilization. Similarly, pregnancy rates did not differ significantly after AI with fresh (57.1%; n = 8/14) or cryopreserved sperm (62.5%; n = 5/8). Seven live rhesus macaques were born following AI with fresh sperm, and three live offspring and two ongoing pregnancies were obtained when cryopreserved sperm were used. Cryopreservation of rhesus sperm as presented here would allow for the cost-effective storage of lineages of nonhuman primates with known genotypes. These results suggest that either national or international centers could be established as repositories to fill the global needs of sperm for nonhuman primate research and to provide the experimental foundation on which to explore and perfect the preservation of sperm from endangered nonhuman primates.     

Effect of different diluents on goat semen fertility

Skim milk (SM) is considered to be the most widely employed extender for goat sperm used for artificial insemination (AI). However, the fertilizing life span of sperm stored in milk or milk-based extenders does not exceed 12h. Besides some seminal plasma components, such as a protein fraction from the goat bulbourethral gland secretion (SBUIII), interacts with some milk fractions and inhibits the spermatozoa motility. The aim of this study was to prolong the survival of buck semen and its fertility. Buck ejaculates were diluted to a final concentration of 100x10(6)spermatozoa/ml with three different diluents: SM, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPOL+hyaluronic acid (TEMPOL+HA). At 7h from dilution 42 goats were inseminated with semen diluted with SM (short-term semen) while after storage for 24h, 44 and 45 goats were inseminated with semen diluted with TEMPOL and TEMPOL+HA (long-term storage), respectively. At day 50 from AI the percentages of pregnant goats were 71.4% (30/42) with SM, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, with significant differences between SM and TEMPOL+HA. The kidding rate was 66.7% (28/42) with SM diluent, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, without significant differences among treatment groups. In conclusion, it is possible to maintain good fertility in goats after AI with semen stored for 24h in TEMPOL.     

beta-D-glucuronidase is associated with goat sperm cytoskeleton

Glycosidase activities were detected as detergent-insoluble after sequential extractions of goat sperm with Triton X-100. Seventy percent of total beta-glucuronidase activity was found in the detergent-insoluble fraction. This portion of beta-glucuronidase was resistant to extractions in the presence of 1 M KCl, chaotropic agents, colchicine, or cytochalasin B, being only partially solubilized by 3 M KCl or DNAse I treatment. The treatment with 0.1% sodium deoxycholate was effective, releasing 73% of the enzyme activity. Treating the deoxycholate extract with DNAse I resulted in a change in the elution profile of beta-glucuronidase as judged by gel filtration chromatography. A polyclonal antibody was developed against pancreatic beta-glucuronidase, and the sperm enzyme was strongly inhibited by the IgG fraction of this antibody. Western blot analysis showed that the same protein correspond to both Triton-soluble and insoluble enzyme. Results demonstrate that beta-glucuronidase is tightly bound to the Triton X-100 resistant fraction, suggesting that the enzyme is associated to sperm cytoskeleton. J. Exp. Zool. 289:146-152, 2001.     

Evaluation of assay conditions for the zona-free hamster ova bioassay of boar sperm fertility

The ability to penetrate zona-free hamster ova may be a very useful test of fresh and frozen boar sperm fertility. These studies were designed to optimize assay conditions prior to evaluation of the accuracy of the bioassay in predicting boar sperm fertility. The ability to penetrate zona-free hamster ova was greater in sperm washed on a Percoll gradient than in sperm washed by dilution and centrifugation. Penetrating ability was greater in sperm from the sperm-rich fraction than from the whole ejaculate but did not differ among different aliquots of the sperm-rich fraction and did not decrease when the prewashing interval was increased from 15 to 85 min. Frequency of collection of ejaculates (1, 3, or 5 times per week) did not affect the penetrating ability of the sperm. Penetration rate was greater when sperm were coincubated with zona-free hamster ova at 39°C compared to 37°C. Sperm from an infertile boar had reduced penetrating ability compared to sperm from fertile boars (11% vs 93%, P < .001). These studies suggest that the zona-free hamster ova bioassay may be a useful assessment of fresh boar sperm fertility.     

Comparison of the fertility of cryopreserved stallion spermatozoa with sperm motion analyses, flow cytometric evaluation, and zona-free hamster oocyte penetration

Stallion spermatozoa were cryopreserved in different extenders, and the correlations between laboratory assay results and sperm fertility were determined. Spermatozoa were cryopreserved in 1) a skim milk-egg yolk medium (CO); 2) a skim milk-egg yolk-sugar medium (SMEY); 3) CO after pretreatment with phosphatidylserine+cholesterol liposomes (CO + L); or 4) cooled to 5 degrees C without cryopreservation. The per cycle embryo recovery rates for mares inseminated with spermatozoa frozen in CO, SMEY, CO + L and spermatozoa cooled to 5 degrees C were 47, 42, 45 and 37%, respectively (P>0.05). The fertility rates of the 5 stallions used were 72, 71, 29, 25 and 16%, respectively (P<0.05). The percentage of motile spermatozoa immediately after thawing (42 to 47%) and after preparation for zona-free hamster oocyte penetration assays (27 to 35%) were not different across treatments (P>0.05). The percentages of motile spermatozoa after cryopreservation were not different across stallions (52 to 58%) initially but were different when spermatozoa were treated with 35 microM dilauroylphosphatidylcholine (PC12) to induce the acrosome reaction (17 to 42%; P<0.05). The percentages of viable spermatozoa and viable acrosome-intact spermatozoa ranged from 30 to 57% and 27 to 48%, respectively, across stallions. The percentages of penetrated hamster oocytes ranged from 19% to 55% and from 24% to 72% when spermatozoa were treated with 35 microM and 50 microM PC12, respectively. The number of spermatozoa penetrating each oocyte ranged from 0.21 to 1.16 sperm/oocyte and from 0.37 to 1.59 sperm/oocyte when spermatozoa were treated with 35 microM and 50 microM PC12, respectively. Analyses of single sperm parameters were not highly correlated with stallion fertility. However, a model utilizing data from flow cytometric analyses (percentage of viable spermatozoa), the percentage of motile spermatozoa, and hamster oocyte penetration (percentage of penetrated hamster oocytes) was highly correlated with stallion fertility (r = 0.85; P = 0.002).     

Motility and fertility of the subtropical freshwater fish streaked prochilod (Prochilodus lineatus) sperm cryopreserved in powdered coconut water

Streaked prochilod (Prochilodus lineatus) is a freshwater fish inhabiting many South American rivers. The objective was to determine the effectiveness of coconut water (ACP™), combined with methylglycol, as a freezing medium for streaked prochilod sperm. A secondary objective was to compare a computer-assisted sperm analyzer (CASA) system versus subjective microscropic examination as a means of assessing sperm motility. As a control, glucose and methylglycol was used, according to our previous study. Sperm diluted in each medium was loaded into 0.5 mL straws, frozen in liquid nitrogen vapor (in a dry shipper), and stored in liquid nitrogen (-196 °C). Half of the samples were evaluated for sperm motility, both subjectively and with CASA; the remainder were evaluated for fertility. There was no difference (P > 0.05) between subjective or CASA assessment of post-thaw sperm motility. Although sperm motility was higher in sperm cryopreserved in ACP™ (85%) than in glucose (75%), cryopreservation in either extender yielded similar fertilization rates (46-48%) and sperm velocities. There were positive correlations (r = 0.56-0.8) between all sperm velocities and fertilization rate. In conclusion, streaked prochilod sperm cryopreserved in glucose or ACP™ and methylglycol was fertile, and thus could be used for research or commercial settings. Furthermore, although the CASA system provided objective data regarding sperm motility, in the present study, subjective evaluation of sperm motility was practical and a good indication of sperm quality; it could readily be done by well-trained personnel under field or laboratory conditions.