Use of the flavonoid taxifolin for sperm cryopreservation from the threatened Bermeya goat breed.

Taxifolin is a plant flavonoid effective as an antioxidant. This study aimed to assess the effect of adding taxifolin to the semen extender during the cooling period before freezing on the overall post-thawing sperm variables of Bermeya goats. In the first experiment, a dose-response experiment was performed with four experimental groups: Control, 10, 50, and 100 µg/ml of taxifolin using semen from 8 Bermeya males. In the second experiment, semen from 7 Bermeya bucks was collected and extended at 20 °C using a Tris-citric acid-glucose medium supplemented with different concentrations of taxifolin and glutathione (GSH): control, 5 µM taxifolin, 1 mM GSH, and both antioxidants. In both experiments, two straws per buck were thawed in a water bath (37 °C, 30 s), pooled, and incubated at 38 °C. Motility (CASA) was assessed at 0, 2, and 5 h, and sperm physiology was assessed at 0 and 5 h by flow cytometry (viability, intact acrosome membrane, mitochondria membrane potential, capacitation, intracellular reactive oxygen species -ROS-, mitochondrial superoxide, and chromatin status). In experiment 2, an artificial insemination trial (AI) was included with 29 goats for testing the taxifolin 5-µM treatment on fertility. Data were analyzed with the R statistical environment using linear mixed-effects models. In experiment 1 and compared to the control, T10 increased progressive motility (P < 0.001) but taxifolin decreased total and progressive motility at higher concentrations (P < 0.001), both post-thawing and after the incubation. Viability decreased post-thawing in the three concentrations (P < 0.001). Cytoplasmic ROS decreased at 0 and 5 h at T10 (P = 0.049), and all doses decreased mitochondrial superoxide post-thawing (P = 0.024). In experiment 2, 5 µM taxifolin or 1 mM GSH (alone or combined) increased total and progressive motility vs. the control (P < 0.01), and taxifolin increased kinematic parameters such as VCL, ALH, and DNC (P < 0.05). Viability was not affected by taxifolin in this experiment. Both antioxidants did not significantly affect other sperm physiology parameters. The incubation significantly affected all the parameters (P < 0.004), overall decreasing sperm quality. Fertility after artificial insemination with doses supplemented with 5 µM taxifolin was 76.9% (10/13), not significantly different from the control group (69.2%, 9/13). In conclusion, taxifolin showed a lack of toxicity in the low micromolar range and could benefit goat semen cryopreservation.

Inhibiting diacylglycerol acyltransferase-1 reduces lipid biosynthesis in bovine blastocysts produced in vitro.

Diacylglycerol acyltransferase-1 (DGAT1) is one of the DGAT enzymes that catalyzes the final step in the synthesis of triacylglycerol, which is a major component of the lipid droplets in embryos. Intracellular lipids accumulated in embryos produced in vitro have been associated with reduced cryotolerance and quality. The objective of the present study was to investigate the influence of DGAT1 inhibition on embryo development, quality, and post-vitrification survival, in addition to expression profiles of selected lipid metabolism-regulating and oxidative stress genes. Bovine cumulus-oocyte complexes were matured and fertilized in vitro and were cultured in synthetic oviduct fluid (SOF) supplemented with 5% fetal calf serum (FCS) alone (Control) or with 1, 5, 10 or 50 µM DGAT1 inhibitor (A922500®; D1, D5, D10, and D50, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for DGAT1 inhibitor dilution) from 54 h post-insemination until Day 8 post insemination. No differences were found in blastocyst yield on days 7 and 8 in Control, CDMSO, D10, and D50 groups. Embryos cultured with 10 or 50 µM DGAT1 inhibitor had greater mitochondrial activity (P < 0.01), and increased number of cells (P < 0.05), while the cytoplasmic lipid content was reduced (P < 0.01), the latter associated with altered expression profiles of selected genes regulating lipid metabolism or genes related with oxidative stress (transcript abundance increased for SLC2A1 and SLC2A5 and decreased for DGAT1 and GPX1). Importantly, the survival rate of blastocysts produced with 10 µM DGAT1 was higher than that of Control, CDMSO and D50 groups at 72 h after vitrification and warming (73.8 vs 57.1, 55.9 and 56.1%, respectively, P < 0.001). In conclusion, inhibition of DGAT1 synthesis in bovine embryos produced in vitro abrogates the negative effect of FCS by decreasing their lipid content, increasing mitochondria activity and improving embryo cryotolerance, as well as favoring the expression of lipid metabolism regulating and oxidative stress-related transcripts.

Evaluation of sperm subpopulation structure in relation to in vitro sperm-oocyte interaction of frozen-thawed semen from Holstein bulls.

The present study examined the relationship between the relative amount of high motile sperm and sperm-oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes.

A comparative study of the sperm nuclear morphometry in cattle, goat, sheep, and pigs using a new computer-assisted method (CASMA-F).

This study was designed to compare the sperm nuclear morphometry of four species of domestic artiodactyls (cattle, sheep, goats, and pigs), using the newly developed automatic computer-assisted sperm morphometry analysis-F. The study was divided into two experiments. In the first experiment, samples from 20 males from each species were collected, diluted, and divided into four sample aliquots. The first was labeled directly with Hoechst 33342, and the others were processed as smears. Between smears, one group was directly labeled with Hoechst after air drying, and the other was fixed either with glutaraldehyde (GLUT), or with methanol, and afterward labeled with Hoechst. Digital images of the fluorescence-labeled sperm were recorded with a digital camera, and at least 200 sperm cells per sample were processed using the Image J analysis open software. Air-drying significantly reduced nuclear sperm dimensions in ruminant species, whereas no effect was observed in pigs. For most of the primary morphometric parameters, the relationship between the four species for the sperm nuclear dimensions can be described as follows: bull > ram ≥ boar > goat. However, ram sperm nuclei had greater width than those of the other species studied. For the secondary morphometric parameters, ram sperm nuclei were clearly less elliptical and elongated and showed greater regularity than in the other studied species. In the second experiment, ejaculates from 10 males per species were used to compare the sperm head morphometric results obtained with the computer-assisted sperm morphometry analysis-F system (using the GLUT treatment as reference) to a more conventional CASMA method (semen smears stained with Harris's hematoxylin and processed with the Integrated Sperm Analysis System [ISAS] commercial software [Proiser R&D SL, Buñol, Spain]). Spermatozoa displayed a bigger size when processed with Harris's hematoxylin than with the GLUT method in all primary sperm head morphometric parameters for the four species studied. A significant correlation was observed between the two methods used in this experiment for all morphometric size parameters in the four species studied. It was concluded that drying and fixation has little effect on sperm nuclear morphometry, with differences between species, and that there are significant variations in size of the sperm nucleus and in the hydrodynamic properties between the four species studied.

Effects of cryopreservation on the motile sperm subpopulations in semen from Asturiana de los Valles bulls.

The aim of this study was to identify different motile sperm subpopulations in ejaculates from an autochthonous bull breed (Bos taurus) and to determine possible modifications in these subpopulations resulting from cryopreservation. Ejaculates were collected and cryopreserved following a conventional protocol. The overall sperm motility and the kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates, after 4h at 5 degrees C, and at 0 and 2h postthaw. A multivariate clustering procedure separated 23,585 motile spermatozoa into four subpopulations: Subpopulation 1 showed medium velocity (VCL: 99.4+/-17.8 microm/sec) and high progressiveness (LIN: 65.1+/-14.0%); Subpopulation 2 included spermatozoa with high velocity (VCL: 148.7+/-25.6 microm/sec) but a nonprogressive trajectory (LIN: 33.1+/-10.5%); Subpopulation 3 represented slowly motile (VCL: 58.3+/-24.3 microm/sec) and nonprogressive sperm (LIN: 39.6+/-18.3%); and Subpopulation 4 included very rapid (VCL: 152.8+/-25.7 microm/sec) and highly progressive sperm (LIN: 70.9+/-13.7%). Subpopulation 4 was present in the greatest quantity in fresh ejaculates (36%), but after cooling, it significantly decreased (21%) concomitantly with an increase (P<0.001) in Subpopulation 2 (from 21% in fresh to 34% in postcooled semen). After freezing and thawing, the overall sperm motility was reduced, mainly due to Subpopulation 2 decreasing from 34% after cooling to 14% after thawing. Differences among bulls in the frequency distribution of spermatozoa within subpopulations were evidenced after thawing by different proportions of spermatozoa in Subpopulations 2 and 4. The current results indicate that a structure of four sperm subpopulations may be a common characteristic of bovine ejaculates and that the cooling phase of cryopreservation seems to be the determinant of postthaw semen quality.

Serum free embryo culture medium improves in vitro survival of bovine blastocysts to vitrification.

The aim of this study was to examine the effects of co-culture with Vero cells during the in vitro maturation (IVM) and three culture media, B2+5% fetal calf serum (FCS) on Vero cells, synthetic oviduct fluid (SOF)+5% FCS, and SOF+20 gL(-1) bovine serum albumin (BSA), on the developmental competence of the embryos and their ability to survive vitrification/warming. We also tested the effect of morphological quality and the age of the embryo on its sensitivity to vitrification. The IVM system neither affects the embryo development up to Day 7 nor survival rates after vitrification. The culture of embryos in SOF+FCS and in Vero cells+B2 allowed obtaining more Day 6 and Day 7 blastocysts, and a higher % of Day 7 blastocysts vitrified than culture in SOF+BSA. Contrarily, on Day 8, more blastocysts were vitrified in SOF+BSA than in SOF+FCS. Blastocysts quality affected development after vitrification/warming, and Day 7 embryos showed higher survival rates than their Day 8 counterparts. Day 7 blastocysts produced in Vero cells or in SOF+BSA survived at higher rates than those produced in SOF+FCS at 24 and 48 h after warming. Embryo culture with BSA allows obtaining hatching rates after vitrification/warming higher than those obtained after co-culture with Vero cells in B2 and FCS. Moreover, this system provides hatching rates from Day 8 blastocysts comparable to those obtained on Day 7 in Vero cells. Further studies, including embryo transfer to recipients, are needed to clarify factors affecting the freezability of in vitro produced bovine embryos.

Effects of re-infection with Neospora caninum in bulls on parasite detection in semen and blood and immunological responses.

Three bulls with experimentally induced primary infection with Neospora caninum were re-infected intravenously with 10(8) live N. caninum tachyzoites of the NC-1 isolate at 300 days post-infection to investigate the presence of N. caninum in semen and blood, and the associated immune responses. In parallel, three bulls with experimentally induced primary infection with N. caninum and three non-infected bulls were also monitored. Re-infected and infected bulls showed an intermittent presence of N. caninum DNA in semen with a parasite load ranging from 0.1 to 15.6 (mean 4.4) and 0.1 to 11.1 (mean 4.1) parasites/ml, respectively. Re-infected bulls showed significant and persistent serum-specific IgM and IgG antibody responses. Specific IgG levels were detected in seminal plasma of all infected bulls, but the magnitude of the response was significantly higher in re-infected rather than in chronically infected animals. The mean specific IFN-gamma levels in re-infected bulls were significantly increased as early as 3 and 7 days after experimental infection when compared to bulls in other groups. This study showed that the intermittent presence and parasite load of N. caninum in the semen of re-infected bulls is very similar to that reported in chronically infected animals. The protozoa could not be isolated from BALB/c nu/nu mice inoculated with PCR-positive semen samples and inseminated heifers with pooled semen samples did not show seroconversion. Plasma IFN-gamma level seems to be a good indicator of a recent N. caninum infection.

Identification of sperm subpopulations with defined motility characteristics in ejaculates from Holstein bulls: effects of cryopreservation and between-bull variation.

The aims of the present study were: (1) to determine the existence of sperm subpopulations with specific motility characteristics in fresh ejaculates from Holstein bulls, (2) to investigate the effects of semen cryopreservation and post-thaw incubation on the distribution of spermatozoa within the different subpopulations, and (3) to evaluate the existence of between-bull variation in the sperm subpopulations structure of fresh and frozen-thawed semen. Six ejaculates were collected from each of 9 Holstein bulls and cryopreserved following a standard protocol. Overall sperm motility and the individual kinematic parameters of motile spermatozoa, determined using a CASA system, were evaluated before freezing and after 0, 2 and 4h of post-thaw incubation at 37 degrees C. Data from 16,740 motile spermatozoa, defined by VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF, were analysed using a multivariate clustering procedure to identify and quantify specific subpopulations within the semen samples. The statistical analysis clustered all the motile spermatozoa into four separate subpopulations with defined patters of movement: Subpopulation (Subp. 1) moderately slow but progressive spermatozoa (23.2%), (Subp. 2) highly active but non-progressive spermatozoa (16.0%), (Subp. 3) poorly motile non-progressive sperm (35.5%), and (Subp. 4) highly active and progressive sperm (25.3%). Subpopulations 2 and 4 significantly (P<0.01) decreased during cryopreservation and post-thaw incubation (Subp. 2: 21.1%, 18.1%, 8.7% and 5.9%; and Subp. 4: 34.1%, 20.6%, 15.2% and 7.3%, respectively, for fresh, 0, 2 and 4h post-thaw) whereas Subp. 3 significantly (P<0.01) increased (10.7%, 27.2%, 27.2% and 30.7%, respectively, for fresh, 0, 2 and 4h post-thaw). The frequency distribution of spermatozoa within subpopulations was quite similar for the 9 bulls, either in fresh or frozen-thawed semen, and differences among bulls were mainly due to differences in the Subp. 4. Significant correlations (P<0.01) were found between the proportions of spermatozoa assigned to Subp. 4 in the fresh ejaculates and those in frozen-thawed semen after 0 (r=0.473), 2 (r=0.513) and 4h post-thaw (r=0.450). This indicated that the ejaculates with the highest subpopulations of rapid and progressive sperm were also the most resistant to cryopreservation and showed the best post-thaw sperm longevity.

Experimental neosporosis in bulls: parasite detection in semen and blood and specific antibody and interferon-gamma responses.

AIM: To investigate the presence of Neospora caninum in semen and blood, and the development of specific antibody and interferon-gamma (IFN-gamma) responses in experimentally infected bulls. METHODS: Eight bulls were intravenously infected with 10(8) live N. caninum tachyzoites of NC-1 isolate. The presence of N. caninum in semen and blood was assessed using a nested-PCR procedure. PCR-positive semen samples were bioassayed using a BALB/c nu/nu mouse model. Specific anti-N. caninum antibody and IFN-gamma responses were also examined. In parallel, eight seronegative bulls were studied as non-infected controls. All bulls were monitored for 26 weeks. RESULTS: All eight experimentally infected bulls showed N. caninum DNA in their semen and/or blood samples at some time during the course of the study. Parasite load in semen ranged from 0.1 to 14.5 parasites/ml (mean 6.0). N. caninum could not be detected in BALB/c nu/nu mice inoculated with PCR-positive semen samples. A significant increase in mean serum specific IgM antibody response to N. caninum was detected between 10 and 28 days post-infection (p.i.). Serum specific IgG, IgG1, and IgG2 antibody levels in experimentally infected bulls were significantly different after 21, 10, and 14 days p.i. as compared to controls, respectively. Specific anti-N. caninum IgG were detected in seminal plasma from infected bulls and values obtained were different from controls after 25 days p.i. Mean specific IFN-gamma responses in experimentally infected bulls were significantly higher than controls 3 days p.i. CONCLUSIONS: This is the first study to report the presence of N. caninum DNA in the semen and blood of experimentally infected bulls. Our observations indicate an intermittent presence of N. caninum in low numbers in semen and associated with chronic stage of the infection. This study is also the first to report the detection of anti-N. caninum IgG in seminal plasma of experimentally infected bulls.

Intrauterine Neospora caninum inoculation of heifers and cows using contaminated semen with different numbers of tachyzoites.

OBJECTIVE: To investigate the potential of different Neospora caninum tachyzoite doses to infect heifers (experiment 1) and cows (experiment 2) when administered in utero by artificial insemination via contaminated semen. METHODS: In experiment 1, five groups of 5, 7, 8, 9, and 5 cyclic heifers were hormonally synchronized and artificially inseminated with semen containing 0 (A, controls), 10(2) (B), 5 x 10(3) (C), 5 x 10(4) (D), and 5 x 10(5) (E) live N. caninum NC-1 isolate-tachyzoites, respectively. Experimental infection was followed for 100 days. Parasitaemia and specific serum IgG, and interferon-gamma (IFN-gamma) responses were studied. In experiment 2, four groups of 9, 10, 9, and 9 adult multiparous cows with confirmed infertility problems of diverse aethiology were hormonally synchronized and artificially inseminated with semen containing 0 (a, controls), 10(2) (b), 5 x 10(3) (c), and 5 x 10(5) (d) live N. caninum NC-1 isolate-tachyzoites, respectively. Experimental infection was followed for 63 days. Parasitaemia and specific serum IgG responses were studied. RESULTS: In experiment 1, parasitaemia was detected in 1, 2, and 3 heifers from groups B, C, and D, respectively, between 9 and 23 days after insemination. Persistent specific serum antibody responses were detected in 2 and 3 heifers from groups D and E, respectively. Transient specific serum antibody responses were detected in 2, 1 and 1 heifers from groups C, D, and E, respectively. In addition, 1 heifer from group B showed a serum-specific antibody level higher than cut off value at 21 days post-insemination. Heifers seroconverted between 23 and 47 days after insemination. Specific IFN-gamma levels were detected in 1, 4, 6, and 3 heifers from groups B, C, D, and E, respectively, between 9 and 55 days after insemination. Pregnancy rate in the control group (60%) was higher than those observed in inoculated heifers (0-42.9%). Pregnancy rates in inoculated heifers were lower when the tachyzoite dose was increased (B 42.9%, C 12.5%, D 11.1%, and E 0%). In experiment 2, no Neospora DNA in blood nor specific serum IgG to N. caninum were detected in any of the cows studied, except in one cow inoculated with 5 x 10(5) tachyzoites (group d) which showed a relative index x100 (RIPC) values of 9.4, 18.9, and 18.1 at 42, 56, and 63 days after insemination, respectively. CONCLUSIONS: This study provides evidence that the intrauterine infection via contaminated semen using 5 x 10(4) and 5 x 10(5) tachyzoites caused persistent serum-specific antibody responses in some heifers. On the basis of serological data, a dose-response effect was also observed. In addition, N. caninum would be a probable cause of early foetal death in inoculated heifers. In contrast, results obtained in a similar experiment with cows showing confirmed infertility indicate that higher doses, such as of 5 x 10(5) tachyzoites, were necessary to induce seroconversion in at least one animal.

Intrauterine Neospora caninum inoculation of heifers.

Here, we studied the potential of Neospora caninum tachyzoites to infect heifers when administered in utero by artificial insemination via contaminated semen. Eighteen primiparous cyclic heifers were hormonally synchronized and artificially inseminated. Nine of them, which were inseminated with semen containing 10(7) live N. caninum NC-1 isolate-tachyzoites, reacted with seroconversion and a specific IFN-gamma response. Moreover, N. caninum DNA was demonstrated by a nested-PCR in the blood of all nine heifers and in brain, lungs, liver and uterine horn of several of them. In contrast, nine heifers inseminated with tachyzoite-free semen developed no antibody or IFN-gamma responses, and no parasite DNA was detected in blood or organs. At necropsy, viable embryos were detected in one and six of the infected and non-infected heifers, respectively. No specific Neospora DNA was detected in any of the embryos. This study provides evidence that intrauterine inoculation via contaminated semen cause N. caninum infection in cattle.

Pregnancies and improved early embryonic development with bovine oocytes matured in vitro with 9-cis-retinoic acid.

Retinoids have an important role in cell growth, morphogenesis and differentiation. In the present study the developmental potential of bovine oocytes was examined after in vitro maturation in the presence of 9-cis-retinoic acid, a vitamin A metabolite, at 5 nmol l(-1) in chemically defined conditions. Experiments studied early in vitro development, blastocyst differential cell counts and the capacity of embryos to establish pregnancy after transfer to recipients. After in vitro fertilization and culture in simple medium, blastocyst development and hatching rates increased in oocytes matured with 9-cis-retinoic acid. Addition of ethanol (used as a solvent for 9-cis-retinoic acid) resulted in higher cell counts and proportions of cells in the inner mass of day 7 blastocysts. Day 8 blastocysts represented most differences observed in the number of cells. In these embryos, ethanol and 9-cis-retinoic acid increased both the number of cells and proportions in the inner mass. However, while ethanol treatment reduced the number of cells in the trophectoderm, 9-cis-retinoic acid did not. The total number of cells was unaffected by treatment within 1 day, although untreated oocytes lead to day 8 blastocysts with reduced total cell counts. Once transferred to recipients, both fresh and vitrified-warmed blastocysts derived from oocytes matured with 9-cis-retinoic acid yielded more pregnancies at day 60. Modifications of retinoid metabolism affect development and trophectoderm differentiation, and in vitro maturation with 9-cis-retinoic acid increased the developmental competence of the oocyte.

Enhancement of developmental capacity of meiotically inhibited bovine oocytes by retinoic acid.

BACKGROUND: Although high vitamin A may be teratogenic to the embryo, retinol has been shown to support oocyte developmental potential in vivo. Similarly, addition of retinol metabolite 9-cis-retinoic acid to in-vitro cultured oocytes could promote cytoplasmic maturation and subsequent early embryonic development. The objective of this study was to evaluate the effects of 5 nmol/l retinoic acid during in-vitro pre-maturation and maturation of bovine oocyte-cumulus complexes. METHODS AND RESULTS: Oocytes were aspirated from cows and either kept under meiotic arrest with 25 micro mol/l roscovitine and matured, or allowed to mature in permissive conditions (control). Cortical granule migration was analysed both after pre-maturation and maturation by fluorescent labelling of oocytes and subsequent laser confocal and fluorescence microscopy. Variables studied after in-vitro fertilization and culture in modified synthetic oviduct fluid were: (i) in-vitro embryonic development; (ii) ability of blastocysts to survive vitrification and warming; and (iii) differential cell counts measured by fluorescence microscopy. Although the presence of 5 nmol/l retinoic acid throughout in-vitro maturation was harmful, its presence during pre-maturation alone improved cytoplasmic granular migration, embryonic development, cryopreservation tolerance, total cell numbers and, as a consequence, embryonic quality. CONCLUSIONS: Pre-maturation in the presence of retinoic acid improves cytoplasmic competence of in-vitro matured bovine oocytes. Until more is known of the molecular mechanisms it would be irresponsible to use retinoic acid in maturation of human oocytes, especially in view of the narrow time window and possible species-specific differences in susceptibility and protection of the oocyte from epigenetic influences of retinol.