Massage therapy in the workplace: reducing employee strain and blood pressure.

AIM: Assess the effects of workplace-based massage therapy on physiological and psychological outcomes. METHODS: We used afield experiment in which 28 participants were randomly assigned into either an experimental (n = 14) or control (n = 14) group. The experimental group received weekly massage treatments at work for a four week period while the control group did not. RESULTS: Both strain and blood pressure were significantly reduced during treatment for the experimental group but not for the control group. CONCLUSIONS: This study provides initial support for the effectiveness of workplace-based massage therapy as part of a comprehensive workplace health strategy.

The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and non-sorted ram spermatozoa.

In an effort to improve the number of functional spermatozoa following sex-sorting and cryopreservation, the effects on in vitro sperm characteristics of the additives: (i) catalase (pre-sorting); (ii) cholesterol-loaded cyclodextrins (CLCs; pre-sorting); and (iii) seminal plasma (post-thawing) were investigated. For all experiments, spermatozoa (three males, n=3 ejaculates/male) were processed using a high speed flow cytometer before cryopreservation, thawing and incubation for 6h. Catalase had no effect (P>0.05) on post-thaw motility characteristics (as measured by CASA) of sex-sorted ram spermatozoa, but pre-sort addition of CLCs reduced (P<0.05) sperm quality after post-thaw incubation for 0 h (motility), 3h (motility, average path velocity, viability and acrosome integrity) and 6h (motility, average path and curvilinear velocity, straightness, linearity, viability and acrosome integrity). Seminal plasma had a differential effect (P<0.001) on sex-sorted and non-sorted spermatozoa. Post-thaw supplementation of increasing levels of seminal plasma caused all motility characteristics of sex-sorted, frozen-thawed spermatozoa to decline (P<0.05); conversely, non-sorted, frozen-thawed spermatozoa exhibited improvements (P<0.05) in motility, viability, acrosome integrity and mitochondrial respiration. In summary, incorporation of catalase, CLCs and seminal plasma into the sorting protocol failed to improve post-thaw sperm quality and, consequently efficiency of sex-sorting of ram spermatozoa. The paradoxical effect of seminal plasma supplementation on the in vitro characteristics of ram spermatozoa provides further evidence that sex-sorting by flow cytometry produces a selected population of cells with different functions compared with non-sorted spermatozoa.

Integration of sperm sexing technology into the ART toolbox.

Sex-sorting of mammalian spermatozoa has applications for genetic improvement of farm animals, in humans for the control of sex-linked disease, and in wildlife as a captive management strategy and for the re-population of endangered species. Considerable research has been undertaken worldwide on the Beltsville sperm sexing technology, the only effective method for pre-selection of sex of offspring. The combination of this method with assisted reproductive technologies has resulted in the birth of offspring in a wide range of animals, including cattle, the only livestock species in which sperm sexing is used commercially. Major improvements in the efficiency of sorting, in particular the development of high speed sorting (15 million X and Y spermatozoa per hour) have led to the production of offspring using conventional and low dose AI and the successful cryopreservation of sorted spermatozoa in cattle, sheep, horses and elk. A major limitation remains the short viable lifespan of sorted spermatozoa in the female genital tract, in most species necessitating sperm deposition deep in the uterus, and close to the expected time of ovulation, for acceptable fertility after in vivo insemination. Special deep uterine insemination technology has been employed to produce offspring in pigs and horses using low sperm doses. Considerable attention has been paid to reduction of the damage and capacitation-like changes to spermatozoa that result from flow cytometric sorting and from freezing and thawing. However, high-purity sorting of liquid-stored or frozen-thawed spermatozoa for immediate use, or re-cryopreservation for later use, does not reduce its fertilizing capacity in vitro, allowing its combination with in vitro fertilization or juvenile in vitro embryo transfer to produce blastocysts, and offspring in sheep and cattle after embryo transfer. Further research into sorting and preservation methods that incorporate strategies to prevent destabilization of sperm membranes may improve the fertilizing lifespan of flow cytometrically sorted spermatozoa. With continued improvement in sorting instrumentation and biological handling, sorting efficiency should reach a point where commercially acceptable pregnancy rates may be achieved in a number of species after conventional or deep uterine insemination.

Liquid storage of flow cytometrically sorted ram spermatozoa.

This study investigated the optimum short-term storage conditions for ram spermatozoa before and after flow cytometric sorting. Prior to sorting, semen from four rams (n = 3 ejaculates per ram) was diluted in either a Tris-based diluent (TRIS) or AndroHep (AH) and stored at 5, 15 or 21 degrees C for 0, 6 or 24h. Sperm characteristics were assessed during storage and after sorting, freeze-thawing and incubation (6h, 37 degrees C). Functional capacity and migration ability in artificial cervical mucus (sperm migration test (SMT)) of stored, sorted and non-sorted (control) spermatozoa were assessed after freeze-thawing. After sorting, semen from three rams (n = 3 ejaculates per ram) was diluted in four different extenders: ultra-heat-treated (UHT) long life milk, TRIS containing 10% (v/v) egg yolk (TRIS-EY), AH (pH 7.4), or TEST buffer containing 10% (v/v) egg yolk (TYB). Sorted and non-sorted (control) spermatozoa were stored at 15 degrees C for 24h or 5 degrees C for 6 days. Sperm characteristics were evaluated at 0, 6 and 24h for samples stored at 15 degrees C and daily for samples stored at 5 degrees C. The SMT was performed on sorted and non-sorted (control) spermatozoa after 6h and 3 days storage at 15 and 5 degrees C, respectively. Spermatozoa stored in TRIS were sorted more efficiently, had higher motility after sorting, freezing, thawing and incubation and had greater numbers of spermatozoa penetrating into the SMT than spermatozoa stored in AH prior to sorting. Spermatozoa stored in UHT at both temperatures had higher motility, acrosome integrity and traveled greater distances in the SMT than spermatozoa stored in all other diluents. In summary, storage in TRIS at 21 degrees C was optimal for transport of ram spermatozoa to the sorting site, and storage of spermatozoa in UHT diluent (after sorting) preserved sperm viability and migration ability best at both 15 and 5 degrees C.

In vitro and in vivo assessment of functional capacity of flow cytometrically sorted ram spermatozoa after freezing and thawing.

The effect of sex sorting and freeze-thawing on the viability and fertility of ram spermatozoa was investigated in the present study. Non-sorted (control) frozen-thawed spermatozoa had a higher motility and forwards progressive motility (FPM) than sorted frozen-thawed spermatozoa (60.9 +/- 2.9% v. 57.0 +/- 3.3% and 4.0 +/- 0.1 v. 3.5 +/- 0.1 FPM, respectively; P < 0.001) after incubation (6 h at 37 degrees C). Sorted and non-sorted (control) frozen-thawed spermatozoa had similar acrosome integrity (73.7 +/- 1.8% v. 75.2 +/- 2.1%, respectively) after thawing and incubation. A greater proportion of sorted spermatozoa displayed chlortetracycline staining patterns that were characteristic of capacitation (22.0 +/- 2.8%; P < 0.05) than non-sorted (control) spermatozoa (15.4 +/- 2.6% B pattern) before freezing. Overall, more sorted frozen-thawed spermatozoa showed patterns characteristic of being acrosome reacted (12.8 +/- 0.7%; P < 0.01) and less were uncapacitated (35.5 +/- 0.6%; P < 0.05) than non-sorted (control) frozen-thawed spermatozoa (7.7 +/- 0.8%; and 38.6 +/- 0.6% for AR and F pattern, respectively). Similar numbers of non-sorted (control) and sorted frozen-thawed spermatozoa migrated through artificial cervical mucus after 1 h (76.4 +/- 11.9 v. 73.9 +/- 11.9 spermatozoa, respectively). The distance travelled by the vanguard spermatozoon was also similar (56.9 +/- 7.8 v. 38.6 +/- 5.8 mm for control and sorted spermatozoa, respectively). Sorted and control frozen-thawed spermatozoa displayed a similar pattern of binding to, and release from, an oviduct epithelial cell monolayer (OECM), but sorted frozen-thawed spermatozoa were released more rapidly (P < 0.05) than non-sorted (control) frozen-thawed spermatozoa. The pregnancy rate was higher for ewes inseminated with 100 x 10(6) (commercial control) frozen-thawed spermatozoa (59%) than for 5, 10, 20 and 40 x 10(6) total sorted frozen-thawed spermatozoa (41% overall; P < 0.001). Insemination of 16 x 10(6) resulted in a higher pregnancy rate (31%) than 10(6) (17%; P < 0.05), but was similar to ewes that received 4 x 10(6) sorted frozen-thawed spermatozoa (24%). Time of insemination (54, 58 and 62 h after sponge removal) had no effect on pregnancy rate. Pregnancy in gonadotrophin-releasing hormone-treated ewes was affected by insemination dose (P < 0.05) but not sperm type (sorted and non-sorted) or ram. Pregnancy was higher after insemination of 40 x 10(6) than 5 or 20 x 10(6) non-sorted (control) or sorted frozen-thawed spermatozoa (70%, 33% and 35%, respectively; P < 0.05). Sorted frozen-thawed spermatozoa may have a shorter viability within the female tract than non-sorted frozen-thawed spermatozoa.

The interaction of fresh and frozen-thawed ram spermatozoa with oviducal epithelial cells in vitro.

In order to investigate the interaction of fresh and frozen-thawed spermatozoa with oviduct epithelial cells, spermatozoa were co-incubated with ovine oviduct epithelial cell monolayers (OECM) derived from either complete oviducts, at any stage of the oestrous cycle (Experiments 1 and 2), or from different regions of the oviduct at different stages of the cycle (Experiment 3). Fresh and frozen-thawed spermatozoa displayed different patterns of binding to, and release from, the OECM. Frozen-thawed spermatozoa immediately bound to the complete oviduct OECM and were released after 2 h. A small proportion of fresh spermatozoa bound immediately, increasing to a maximum after 2 h, and were gradually released thereafter. When only the cells that were released from the OECM were observed by chlortetracycline staining in Experiment 2, it was found that the presence of an OECM increased the number of capacitated fresh spermatozoa while decreasing the number of capacitated frozen-thawed spermatozoa. Overall, the OECM advanced the membrane state of both types of spermatozoa from uncapacitated to acrosome-reacted. Fresh and frozen-thawed spermatozoa bound to OECM derived from the cells of the isthmus and the ampulla in similar proportions. However, more spermatozoa were capacitated when incubated with OECM derived from isthmic rather than ampullary cells. Higher proportions of fresh spermatozoa bound to, and were acrosome-reacted following incubation with OECM derived from post- rather than pre-ovulatory tracts. Such differences were not observed for frozen-thawed spermatozoa. The findings reported in this study show that fresh and frozen-thawed spermatozoa behave differently when in contact with oviduct cells in vitro. This may be a consequence of the more advanced membrane state of the frozen spermatozoa upon thawing.

Fate and functional integrity of fresh and frozen-thawed ram spermatozoa following intrauterine insemination.

Ewes in a synchronized oestrus were inseminated (intrauterine) with fresh and frozen-thawed spermatozoa and the spermatozoa were either recovered from each section of the reproductive tract after the animal was killed (Experiments 1a and 1b) or after they were voided from the cervix (Experiment 2). In Experiment 1a, only 1.2+/-0.27% of the original inseminate was recovered. Placing a ligature at the base of the uterine horn in Experiment 1b led to the recovery of 3.0+/-0.33% of the original inseminate, located mainly in each uterine horn (33.1+/-5.48%), and each isthmic and ampullary region of the oviduct (2.9+/-5.48% and 4.0+/-5.48%, respectively). A higher proportion of spermatozoa recovered from the isthmus were uncapacitated when observed by chlortetracycline staining than those recovered from the uterus (26.4+/-1.92% and 15.6+/-1.92%, respectively, P<0.05). Experiment 2 showed that large proportions of spermatozoa were voided from the tract through the vagina, with similar numbers of fresh and frozen-thawed spermatozoa lost from the tract. However, frozen thawed spermatozoa were lost at a faster rate than fresh (P<0.05) and with a more advanced membrane state (66.8+/-1.30% and 53.2+/-1.30% were acrosome reacted respectively; P<0.001). Large numbers of recovered spermatozoa had lost their tails, with frozen-thawed spermatozoa more susceptible to tail loss than fresh spermatozoa (55.0+/-0.96% and 45.5+/-0.96% respectively; P<0.05).

Normal fertility in ewes after cervical insemination with frozen-thawed spermatozoa supplemented with seminal plasma.

The effect of seminal plasma (SP) on the motility, capacitation status, penetration through cervical mucus and fertility of frozen-thawed ram spermatozoa was examined. In the presence of SP, motility of frozen-thawed spermatozoa was better (P<0.001) and there were more uncapacitated and less acrosome-reacted cells in comparison with controls (P<0.001). Frozen thawed spermatozoa were also better able to penetrate cervical mucus after addition of SP. Addition of SP increased the percentage of ewes pregnant after insemination of frozen-thawed (39/94, 41.5% v. 51/92, 55.4%; P<0.05) but not fresh spermatozoa (34/55, 61.8% v. 42/58, 72.4% for 0 v. 30% SP in the resuspension medium). Moreover, SP improved pregnancy rates after cervical (14/50; 28% v. 25/49; 51%; P<0.05) but not intrauterine insemination (25/44; 56.8 v. 26/43; 60.5%) with frozen-thawed spermatozoa. In a second experiment, pregnancy rates were 30/45 (66.7%), 9/37 (24.3%) and 24/40 (60.0%) for ewes inseminated with frozen-thawed spermatozoa in the uterus (control), cervix without SP and cervix after supplementation with SP, respectively (P<0.01 for unsupplemented v. supplemented spermatozoa). These experiments demonstrate that impaired function of cryopreserved spermatozoa can be overcome by addition of SP, resulting in normal fertility after cervical AI.

The functional integrity and fate of cryopreserved ram spermatozoa in the female tract.

Cryopreservation advances capacitation-like changes in ram spermatozoa. These changes are reflected in an increased fertilizing ability compared with fresh spermatozoa, followed by an accelerated decline in fertilizing ability after incubation in vitro or in vivo. Furthermore, frozen-thawed spermatozoa are released earlier than fresh spermatozoa after binding to oviduct cells in vitro, confirming their physiological readiness to participate in fertilization despite their short lifespan. After insemination large numbers of spermatozoa are lost from the female reproductive tract of the ewe via the vagina. Frozen-thawed spermatozoa are expelled faster than fresh spermatozoa. The advanced membrane status of frozen-thawed spermatozoa may provoke their rapid loss and possibly makes them more vulnerable to attack by uterine leucocytes, or by some other mechanism, as a high proportion of spermatozoa lost from the tract are decapitated. The observed destabilization of the membranes of cryopreserved spermatozoa is accompanied by impaired sperm transport, associated with mitochondrial injury, necessitating intrauterine deposition of frozen-thawed semen to obtain satisfactory fertility after artificial insemination. However, the frozen-thawed spermatozoa that can participate in fertilization may contribute to increased embryonic loss by the advancement of cleavage or through a direct effect of cryopreservation on the male genome.

Time course of pronuclear formation and fertilisation after insemination in vitro and intracytoplasmic sperm injection of in vitro matured sheep oocytes.

The time course of sperm decondensation, oocyte activation, pronuclear formation and the possible causes of abnormalities after intracytoplasmic sperm injection (ICSI) and in vitro fertilisation (IVF) were examined. Frozen-thawed and pooled fresh semen from three different rams were washed and capacitated for ICSI or IVF. In vitro matured oocytes were cultured after sperm injection for 0.5, 0.75, 1, 2, 3, 4, 5, 6, 8, 18, 21 and 23 h, and oocytes were cultured after in vitro insemination for the same times other than 18 and 23 h. All oocytes were cultured in bicarbonate-buffered synthetic oviduct fluid medium (BSOF) supplemented with 2% oestrous sheep serum. A total of 746 metaphase II oocytes were injected with a single spermatozoon and 986 oocytes were inseminated for IVF. The earliest oocyte activation after ICSI was observed at 0.5 h, when 14.8% of oocytes were in anaphase II; this was earlier than after IVF, when only 6.4% of the oocytes exhibited anaphase II 1 h after insemination. Decondensing spermatozoa were first observed 1 h after ICSI and 3 h after insemination for IVF. The earliest female and male pronuclei after ICSI were observed at 2 and 3 h respectively, while the female and male pronuclei after IVF were observed at 4 h after insemination. The overall fertilisation rate was lower after ICSI (28.6%) than IVF (70.4%) but the percentage of abnormal fertilisation was not different between ICSI (8.7%) and IVF (15.2%). It was concluded that the fertilisation events were more advanced for ICSI than IVF, using injection and insemination time as reference points. The formation of male and female pronuclei were asynchronous after ICSI, in contrast to IVF when they appeared simultaneously at 4 h. Abnormalities found in fertilisation after ICSI may therefore be induced by the injection technique.

Effect of culture, incubation and acrosome reaction of fresh and frozen-thawed ram spermatozoa for in vitro fertilization and intracytoplasmic sperm injection.

This study evaluated different sperm treatments for fertilization of sheep oocytes by intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF). In Experiment 1, fresh and frozen semen was separated by Percoll centrifugation and incubated at 30 degrees C or 39 degrees C in HSOF or BSOF medium for 1 h before use for IVF or ICSI. For IVF, oocytes were inseminated and incubated with sperm for 30 min, 4 h and 19 h. Sperm were assessed for acrosome integrity after Percoll centrifugation and 1 h incubation, and those used for IVF were assessed after each period of exposure to the oocytes. Fertilization rates after ICSI were higher for fresh than for frozen-thawed sperm and were highest 19 h after IVF with fresh or frozen-thawed sperm in the presence of HSOF at 30 degrees C. In Experiment 2, fresh semen was separated by Percoll centrifugation and incubated for 5 h in HSOF, and the acrosome reaction was induced with lysophosphatidylcholine. Acrosome integrity was then assessed. Fertilization rates after ICSI were similar for acrosome-reacted and control spermatozoa. These results suggest that induction of the acrosome reaction in spermatozoa before ICSI is unnecessary, whereas a capacitating treatment of spermatozoa is required before IVF.

Capacitation status and fertility of fresh and frozen-thawed ram spermatozoa.

The effect of cryopreservation on the capacitation status and fertility of ram spermatozoa was observed. After the chlortetracycline staining technique was validated for ram spermatozoa, it was applied to fresh or long-term frozen-stored spermatozoa. Fresh spermatozoa displayed mainly the F pattern (non-capacitated; 61.3%), becoming B pattern (capacitated; 54%) and AR pattern (acrosome reacted; 41%) with incubation (6 h at 37 degrees C). In contrast, frozen spermatozoa displayed the B pattern (65.9%), becoming the AR pattern (64.2%) with incubation. This demonstrates that cryopreservation may cause membrane changes in ram spermatozoa functionally equivalent to capacitation. The differences in capacitation status did not affect in vitro fertilization rates between fresh and frozen spermatozoa, but pregnancy rates at Day 18 after intrauterine artificial insemination were higher for fresh than for frozen spermatozoa. This difference was not evident at Day 50, possibly as a result of the high embryonic loss between Days 18 and 50 when fresh unincubated and frozen incubated spermatozoa were inseminated. Further research is necessary to determine what part of the cryopreservation process is responsible for the membrane changes in ram spermatozoa.

Glycerol 3-phosphate dehydrogenase of boar spermatozoa: inhibition by alpha-bromohydrin phosphate.

Boar spermatozoa incubated with glycerol 3-phosphate as substrate produced CO2 and an accumulation of dihydroxyacetone phosphate and fructose-1,6-bisphosphate. The rate of oxidation of glycerol 3-phosphate was decreased, as was the production of CO2 and the two glycolytic intermediates, in the presence of (R,S)-alpha-bromohydrin phosphate. In the presence of inhibitors of stage two of the glycolytic pathway, CO2 production was prevented, there was a marked increase in the concentration of the glycolytic intermediates but the rate of metabolism of the substrate was unaffected. Oxygen consumption by spermatoza incubated with glycerol 3-phosphate was unaffected in the presence of rotenone, whereas it was decreased when lactate was offered as the substrate. The results reported here confirm that in boar spermatozoa glycerol 3-phosphate dehydrogenase is an FAD-linked enzyme that is inhibited by (R,S)-alpha-bromohydrin phosphate in, possibly, a competitive manner.

Metabolism of glycerol 3-phosphate by mature boar spermatozoa.

Under anaerobic conditions boar spermatozoa metabolized fructose and glucose to lactate but did not produce ATP to the extent of that produced under aerobic conditions; the ketogenic amino acids leucine, tryptophan, phenylalanine and tyrosine were not oxidatively metabolized. Glycerol 3-phosphate was metabolized rapidly in the presence or absence of the glycolytic inhibitor, 3-chloro-1-hydroxypropanone (CHOP). In the absence of CHOP, glycerol 3-phosphate was converted to CO2, lactate, glucose 6-phosphate and fructose 6-phosphate, and ATP was produced. In the presence of CHOP, glycerol 3-phosphate did not produce CO2, lactate or ATP, but formed fructose 1,6-bisphosphate and dihydroxyacetone phosphate. With dihydroxyacetone phosphate as substrate, fructose 1,6-bisphosphate, lactate, glucose 6-phosphate, fructose 6-phosphate and ATP were produced. Accumulation of glucose 6-phosphate and fructose 6-phosphate from glycerol 3-phosphate appeared to depend on the production of ATP; if ATP was not produced, dihydroxyacetone phosphate and fructose 1,6-bisphosphate accumulated. The conversion of glycerol 3-phosphate to glycolytic intermediates appeared to be a mechanism for the conversion of substrates for the ultimate production of lactate.

The anti-glycolytic activity of 3-bromopyruvate on mature boar spermatozoa in vitro.

Mature epididymal boar spermatozoa converted fructose, glycerol and glycerol-3-phosphate to carbon dioxide, but in the presence of 0.5 mM 3-bromopyruvate, these oxidations were inhibited while that of lactate was unaffected. Inhibition of the oxidation of these substrates results in a decrease in the content of ATP and the accumulation of dihydroxyacetone phosphate and fructose-1,6-bisphosphate. Examination of the activities of the enzymes within stage two of the glycolytic pathway showed that glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase were immediately inhibited by 3-bromopyruvate in a competitive manner. We now report the action of 3-bromopyruvate on the metabolic activity of boar spermatozoa. At a concentration of 0.5 mM, this compound selectively inhibits stage two of the glycolytic pathway and becomes yet another specific inhibitor of spermatozoal metabolism.

Amniotic fluid alpha-fetoprotein subfractions in fetal Trisomy 21 affected pregnancies.

The percentage contribution to the total AFP concentration in amniotic fluid from the yolk sac derived AFP was measured at 15-17 weeks gestation in 15 pregnancies affected by fetal Trisomy 21 and compared with that in 75 pregnancies with a normal outcome. The yolk sac form of AFP was reduced in parallel to the total AFP and differentiation of the subfraction from the yolk sac would offer no increase in diagnostic efficiency.