Crystal structure of 1-ferrocenyl-2-(4-nitro-phen-yl)ethyne.

The title ferrocene derivative, [Fe(C5H5)2(C8NO2)], including an alkyne bonded to a para-nitro-phenyl substituent, which was synthesized from a copper-free Sonogashira cross-coupling reaction between ethynylferrocene and 4-bromo-1-nitro-benzene, crystallizes in the P21/n space group. In the ferrocene unit, the penta-dienyl (Cps) rings are in an eclipsed conformation. The angle of rotation between the substituted cyclo-penta-dienyl ring and the p-nitro-phenyl group is 6.19 (10)°, yielding a quasi-linear extension of the ferrocenyl substitution. Important inter-molecular inter-actions arise from π-π stacking between the Cp rings and the p-nitro-phenyl, from corners of the Cp rings that are perpendicularly aligned, and between the O atoms from the nitro substituent and carbons at the corners of the Cp rings, propagating along all three crystallographic axes.

Influence of sperm filtration and the addition of glycerol to UHT skimmed milk- and TEST-based extenders on the quality and fertilizing capacity of chilled ram sperm.

The poor fertility of ram semen stored chilled for long periods has encouraged the development of protocols designed to improve the kinetic vigour and cervical barrier-crossing capacity of sperm. The present work evaluated the effect of sperm selection with Sephadex filtration and the supplementation of 2% glycerol (GLY) to extenders based on ultra-heat-treated skimmed milk (UHT) or Tris-Tes-Glucose (TEST) on ram sperm kinetic parameters, plasma membrane integrity, acrosome integrity, mitochondrial function and fertilizing ability, over long chilling times. The results showed that for non-filtered semen, values for progressive sperm motility (%PSM), straight line velocity (VSL, µm/s) and the percentage of sperm with an intact plasma membrane/intact acrosome/a high mitochondrial function index (%IPIAHM) at all times up to 96 h of chilling were higher when the UHT extender (P < 0.01) was used compared to TEST extender irrespective of the presence of GLY. When semen was previously filtered with Sephadex, the addition of GLY to the UHT extender improved total motility (%TM), the %PSM and the VSL at 96 h compared to all other treatments (P < 0.01). The best results of all were obtained with non-filtered semen and UHT either with or without GLY. Heterologous IVF using zona-intact bovine oocytes was used to assess the fertilizing capacity of non-filtered fresh (FS0), chilled-for-24 h (CS24) or chilled-for-48 h (CS48) ram semen diluted in UHT extender (GLY-free). Heterologous IVF showed that ram sperm, either FS0, CS24 or CS48, were equally capable of penetrating zona pellucida intact bovine oocytes, leading to pronuclear formation and hybrid embryo cleavage (46.3 ±â€¯3.2; 48.8 ±â€¯3.2; and 43.3 ±â€¯3.5, respectively). No differences were seen with respect to fresh sperm in terms of sperm binding, penetration, polyspermy, pronucleus formation or cleavage rates (P > 0.05). In conclusion, neither Sephadex filtration nor addition of glycerol provided extra benefits to ram sperm chilled up to 96 h. Chilled, non-filtered sperm extended with UHT without GLY showed better sperm functionality than did similar sperm extended with TEST extenders. Indeed, sperm diluted in UHT extender, maintained fertilizing ability up to 48 h.

Effect of shortening the prefreezing equilibration time with glycerol on the quality of chamois (Rupicapra pyrenaica), ibex (Capra pyrenaica), mouflon (Ovis musimon) and aoudad (Ammotragus lervia) ejaculates.

The present study reports the effect of shortening the prefreezing equilibration time with glycerol on the quality of frozen-thawed ejaculated sperm from four Mediterranean mountain ungulates: Cantabrian chamois (Rupicapra pyrenaica), Iberian ibex (Capra pyrenaica), mouflon (Ovis musimon) and aoudad (Ammotragus lervia). Ejaculated sperm from these species were divided into two aliquots. One was diluted with either a Tris-citric acid-glucose based medium (TCG-glycerol; for chamois and ibex sperm) or a Tris-TES-glucose-based medium (TTG-glycerol; for mouflon and aoudad sperm), and maintained at 5°C for 3h prior to freezing. The other aliquot was diluted with either TCG (chamois and ibex sperm) or TTG (mouflon and aoudad sperm) and maintained at 5°C for 1h before adding glycerol (final concentration 5%). After a 15min equilibration period in the presence of glycerol, the samples were frozen. For the ibex, there was enhanced (P<0.05) sperm viability and acrosome integrity after the 3h as compared with the 15min equilibration time. For the chamois, subjective sperm motility and cell membrane functional integrity were less (P<0.05) following 15min of equilibration. In the mouflon, progressive sperm motility and acrosome integrity was less (P<0.05) when the equilibration time was reduced to 15min. For the aoudad, the majority of sperm variables measured were more desirable after the 3h equilibration time. The freezing-thawing processes reduced the sperm head size in all the species studied; however, the equilibration time further affected the frozen-thawed sperm head variables in a species-dependent fashion. While the equilibration time for chamois sperm might be shortened, this appears not to be the case for all ungulates.

Successful ultrarapid cryopreservation of wild Iberian ibex (Capra pyrenaica) spermatozoa.

A method for cryopreserving wild ibex sperm at high cooling rates was developed. To design a freezing solution based on Tris, citric acid, and glucose (TCG), two preliminary experiments were performed using glycerol (GLY) and dimethyl sulfoxide (DMSO) at different concentrations (5%, 10%, 20%). The 10% GLY + 10% DMSO combination reduced (P < 0.05) frozen-thawed sperm motility, which reached a minimum when 20% GLY + 20% DMSO was used. In the second experiment, sperm tolerance to three sucrose concentrations was evaluated (100-mM sucrose, 300-mM sucrose, 500-mM sucrose). Frozen-thawed sperm motility and sperm viability decreased (P < 0.05) at concentrations above 300 mM. The ultrarapid cooling procedure finally used involved a TCG egg yolk (ey)-based extender with 100-mM sucrose, either alone or with 5% GLY with or without BSA. Two warming procedures (37 °C vs. 60 °C) were also evaluated. The TCG ey with 100-mM sucrose but without GLY/BSA returned the best sperm quality variables. Slow warming at 37 °C strongly affected (P < 0.05) sperm motility and viability in all groups. Sperm selection by density gradient centrifugation produced no motile sperm when slow warming was performed. In contrast, when fast warming was used, sperm selection increased (P < 0.05) percentage of motility, viability, and the percentage of sperms with intact acrosomes. Heterologous in vivo fertilization involving domestic goats was performed to evaluate the in vivo fertilization capacity of the ultrarapidly cooled cryopreserved sperm (in TCG-ey + 100 mM sucrose), with warming undertaken at 60 °C. Inseminations of domestic goats resulted in three pregnancies (3 of 16, 18.7% fertility). In conclusion, ibex spermatozoa are strongly sensitive to high concentrations of permeable cryoprotectants and sucrose. However, the combination of ultrarapid cooling, using TCG-ey + 100-mM sucrose, and fast warming at 60 °C, followed by sperm selection by density gradient centrifugation to collect the motile sperm, has a positive effect on sperm viability.

Cryopreservation of epididymal sperm from ibexes (Capra pyrenaica) using short equilibration time with glycerol.

Two experiments were conducted to study the effect of shortening the equilibration time with the cryoprotectant glycerol before freezing epididymal sperm recovered postmortem from Iberian ibex. In the first experiment, the standard equilibration time of 3 hours was compared with 2 hours, and subjective sperm motility and quality of movement were greater (P < 0.05) in the latter group. In the second experiment, reducing the equilibration time from 2 hours to 15 minutes did not affect sperm motility (evaluated subjectively and objectively), viability, acrosomal integrity, or membrane functional integrity. In conclusion, shortening the equilibration time can be used as a technique to simplify the cryopreservation process and this provides practical advantages under field conditions.

Sperm characteristics and heterologous in vitro fertilisation capacity of Iberian ibex (Capra pyrenaica) epididymal sperm, frozen in the presence of the enzymatic antioxidant catalase.

The aim of this work was to evaluate the protective effect of catalase (CAT) on frozen/thawed ibex epididymal sperm recovered post mortem, and to detect any harmful effect this might have on sperm fertilisation capacity. Epididymal spermatozoa were diluted using a Tris-citric acid-glucose medium (TCG) composed of 3.8% Tris (w/v), 2.2% citric acid (w/v), 0.6% glucose (w/v), 5% glycerol (v/v), and 6% egg yolk (v/v). Sperm masses from the right epididymis were diluted with TCG medium, while those from the left were diluted with TCG medium supplemented with 200IU/mL CAT. Heterologous in vitro fertilisation (IVF) was used to assess the fertilisation capacity of this sperm. The addition of CAT to the extender did not improve frozen/thawed sperm variables. Moreover, a reduced fertilisation capacity was detected: sperm diluted with TCG provided 25.5% 2PN zygotes, while just 13.2% was recorded for that diluted with TCG-CAT (P<0.01). The percentage of cleaved embryos at 48hpi was higher (P<0.01) with the TCG sperm than with the TCG-CAT sperm (16.7% vs. 7.6%). The use of 200IU/mL CAT as an additive cannot, therefore, be recommended for the preservation of ibex epididymal sperm. Other antioxidants should, however, be tested in both this and related wild mountain ungulates.

Efficiency of estrous synchronization in tropical sheep by combining short-interval cloprostenol-based protocols and "male effect".

This study assessed the efficacy of a protocol combining short-interval cloprostenol-based protocols and "male effect" for estrous synchronization in hair sheep. In Experiment 1, 24 ewes were randomly assigned to three groups (n=8) and treated with cloprostenol on Days 3, 5 and 7 after ovulation, respectively. Estradiol secretion during the follicular phase was similar among groups. Onset of estrus (P<0.001) and the timing of maximum LH concentration (P<0.01) were earlier in group D3 than in D5 and D7 groups. During the subsequent cycle, the number and size of corpora lutea were higher (P<0.05) in ewes of the groups D3 (1.9+/-0.3 and 115.1+/-14.3mm(2)) and D5 (1.8+/-0.2 and 100.2+/-11.2mm(2)) than in group D7 (1.3+/-0.2 and 75.6+/-6.4mm(2)) group. In Experiment 2, 24 ewes were treated with two cloprostenol injections (7 days apart). Twelve ewes were exposed to "male effect" previous to an isolation period (ME group), whereas the remaining ewes were controls without male exposure (CTR group). Male effect induced earlier preovulatory LH surge (P<0.05) and ovulation (P<0.001) than CTR group. In Experiment 3, the estrus was synchronized in 68 ewes. Nineteen of them (group FGA) were treated using intravaginal sponges impregnated with fluorogestone acetate for 12 days and inseminated at 55h. Forty-nine females (group ME) were treated like ME group. Twenty-four (ME48 group) and 25 ewes (ME55 group) were inseminated at 48 and 55h after treatment, respectively. The fertility rate was numerically higher in ME48 than ME55 and FGA groups (62.5, 44.0 and 47.4%, respectively). In conclusions, the combined use of short-interval cloprostenol treatment and "male effect" may be an adequate alternative for synchronizing estrus and applying artificial insemination in hair sheep throughout the entire year.

Ovarian and endocrine responses in tropical sheep treated with reduced doses of cloprostenol.

The present study aimed to assess the efficacy of reduced doses of cloprostenol for synchronizing estrus and ovulation in hair sheep. With the aim to evaluate the luteolytic activity of reduced cloprostenol doses, a first experiment was performed using a relatively large (group H: 126 microg; n=8), medium (group M: 68.25 microg; n=6) and small (group L: 38.5 microg; n=6) cloprostenol dose. Luteolysis was assessed at Days 3 and 6 after injection (Day 0) by progesterone concentrations (P(4)) and transrectal ultrasonography (US). In Experiment 2, sheep were randomly assigned to the same three doses to evaluate a protocol for estrous synchronization using two injections administered 9 days apart. A third trial was performed with ewes treated (9 days apart) with the large dose (H=126 microg; n=12) and with a small dose adjusted for facilitating volume management (LA=43.75 microg; n=12). Presence of estrous cycling was determined in all the ewes by US and P(4) assay, at Days -9, -6, -2, 0 (Day of second cloprostenol injection), 8 and 11. Bleeding and US were done every 4h from 16 h of the beginning of the estrus during the third trial to assess the preovulatory LH surge and timing of ovulation. Additionally, blood samples were drawn at Days 0, 1, 2 and 3 to assess estradiol (Experiments 2 and 3) and P(4) (Experiment 2) concentrations during the ovarian follicular phase. In all experiments, percentage of animals showing luteolysis, preovulatory follicular dynamics and function and percentage of ewes showing behavioral estrus in response to treatment was similar among groups. Timing of estrus for group H was earlier than group L (28.6+/-1.8h compared with 37.1+/-2.4h; P<0.05). In the third trial, the preovulatory LH peak was higher in the LA group than group H, in terms of maximum mean concentration during the surge (27.7+/-1.8 ng/mL compared with 21.3+/-2.2 ng/mL; P<0.05) and area under the curve (AUC; 183.4+/-12.7 ng/mL compared with 127.7+/-10.9 ng/mL; P<0.01). However, timing of ovulation was similar for H and LA groups. Thereafter, ovulation rate and luteal function at Day 11 were similar. Current results demonstrate that reduced doses of cloprostenol may be applied in a practical manner for reproductive management of sheep, with the additional advantage of reducing treatment costs.

Systemic and intraovarian effects of corpus luteum on follicular dynamics during estrous cycle in hair breed sheep.

The present study aimed to determine systemic and local effects of corpora lutea (CL), on follicular dynamics throughout the estrous cycle. All follicles >or=2 mm and CL were assessed by daily transrectal ultrasonography in 12 West African ewes. Blood samples were collected to determine plasma concentration of progesterone. Fifteen estrous cycles were evaluated with a mean interovulatory interval of 16.8+/-0.2 days. Two (13.3%), 10 (66.7%) and 3 (20%) of the estrous cycles had 2, 3 and 4 waves of follicular development, respectively. In sheep with three waves of follicular development, both the length of growing phase and the growth rate of dominant follicles from midluteal wave II were diminished (3.4+/-0.3 days, P<0.0001, and 0.4+/-0.1 mm/day, P<0.01, respectively) when compared to follicles from early luteal phase (wave I, 4.1+/-0.2 days, and 0.7+/-0.1 mm/day) or late luteal phase (wave III, 6.3+/-0.4 mm and 0.6+/-0.1 mm/day). The diameter of the dominant follicle was smaller during the midluteal phase (3.9+/-0.1 mm, P<0.0001) than in the early and late luteal phase (5.0+/-0.2 and 5.7+/-0.2 mm; respectively). The effect of the dominant follicle was less during midluteal phase, because number of accompanying smaller follicles was fewer (P<0.01) in waves I and III (6.3+/-0.9 compared with 3.4+/-0.8 and 2.3+/-0.7). The number of follicles was also different between ovaries that had CL and those that did not. The total number of large follicles during the luteal phase was less in ovaries with CL (0.9+/-0.5 compared with 2.7+/-0.3; P<0.01), as was the mean daily number of both large (0.1+/-0.02 compared with 0.2+/-0.02; P<0.001) and total number of follicles >or=2 mm (2.5+/-0.1 compared with 3.3+/-0.1; P<0.01). Current results indicate that the presence of a functional CL may exert both systemic and local effects on the population of follicles, affecting the dominance exerted by large follicles.