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Biomedical subjects

A W Blackshaw

Publications and source records attributed to A W Blackshaw.

At least 19 recordsLinked to original sources
PubMed2006

Cryopreservation and transfer of pig embryos.

Recent advances in cryopreservation and non-surgical transfer of pig embryos have made embryo transfer in pigs a commercially viable technology especially for the international transfer of valuable genetic material. Early research demonstrated that early stage pig embryos were highly sensitive to temperatures below 15 degrees C and this sensitivity decreased with development to peri-hatching blastocysts which is accompanied by a reduction in lipid content. Removal of the lipid prior to freezing was found to improve the freezability and resulted in the birth of piglets. It was shown that by polarising the lipid material out of the blastomeres and using ultra rapid freezing (vitrification) the early stage zona pellucida intact embryo would also survive freezing and result in the birth of piglets. Other factors that have contributed to successful cryopreservation of the early stage pig embryo include the use of cytoskeletal stabilising agents, specific cryoprotectants, increasing cooling rates using open pulled straws or micro droplets and assisted hatching. The present review discusses the relative importance of each of these factors. The development of non-surgical transfer technology and its importance in the application of embryo transfer in the pig industry is also reviewed.

Animals↗
PubMed2005

Changes to porcine blastocyst vitrification methods and improved litter size after transfer.

The objective was to improve the protocol that was used to obtain the first reported piglets from transferred vitrified and warmed zona-intact blastocysts. Blastocysts were collected from superovulated sows and gilts, centrifuged to polarize lipid, vitrified, warmed and cultured for 24h or transferred immediately. Removing the zona pellucida after warming increased the number of cells in the surviving blastocysts (zona-free 60.8+/-4.3, zona-intact 39.1+/-2.8; P<0.05). Thinning the zona pellucida produced similar results to zona removal. Changing the basal medium of the vitrification and warming solutions from modified PBS to phosphate buffered NCSU-23 increased the number of cells (44.7+/-2.2 versus 56.0+/-3.9, respectively; P<0.05). Reducing the plunge temperature of the liquid nitrogen from -196 degrees C to less than -204 degrees C improved the embryo survival rate (61.9% versus 82.9%, respectively; P<0.05). These modifications were incorporated into the vitrification protocol that was used to vitrify and warm 105 blastocysts (that were subsequently transferred into four recipients). Three recipients became pregnant, farrowing three litters (average litter size, 5.3; 18.8% embryo survival in farrowing sows). Changing the warming protocol to using sucrose rather than ethylene glycol resulted in a trend towards improved embryo survival (73.5% versus 91.2%) but this was not statistically significant. Incorporating this modification, 203 blastocysts were vitrified, warmed and transferred into seven recipients. Five became pregnant and 36 fetuses were recovered (average litter size 7.2; 24.8% embryo survival in pregnant sows) at Day 40 of pregnancy. In conclusion, changes made to the vitrification protocol improved pregnancy rate and in vivo embryo survival compared to an earlier study using the original protocol.

Animals↗
PubMed2004

Farrowing rates and litter size following transfer of vitrified porcine embryos into a commercial swine herd.

The objective was to determine farrowing rates and litter sizes that could be achieved in a typical farm-to-farm porcine embryo transfer program using vitrified blastocysts that were zona pellucida intact when cryopreserved. The embryos were transferred surgically on-farm into recipient sows that were managed throughout gestation and farrowing under the same conditions as other sows in the herd. Twenty recipient sows (mean parity 2.1) received a total of 568 embryos; seven received 203 embryos derived from donor sows, five received 139 embryos from gilts and eight received a mixture of 161 embryos from sows and 65 from gilts. Sixteen sows (80%) were confirmed pregnant at approximately 35 days gestation, 15 farrowed at full term (farrowing rate 75%). One sow died during gestation (with a total of 18 fetuses in utero). A total of 123 piglets were born (mean, 8.2), of which 115 were born alive (mean, 7.7). Of the 568 embryos transferred to all 20 sows, 21.6% resulted in piglets born and 29.0% survived to produce piglets in sows that farrowed. There were no significant differences in embryo survival among sow, gilt or mixed sow and gilt embryos. The ratio of males to females was 71/52 and the mean birth weight was 1.6 kg (range 0.6-2.6 kg). In conclusion, vitrified zona pellucida intact embryos can be used to transfer genetic material from farm-to-farm with acceptable reproductive performance.

Animals↗
PubMed2002

Piglets born from centrifuged and vitrified early and peri-hatching blastocysts.

Cryopreservation of zona-intact porcine embryos has been relatively unsuccessful to date, although some success has been obtained with lipid reduced morulae and early blastocysts. This study adapted some vitrification protocols used successfully with late blastocysts for use with early zona-intact blastocysts, using actin depolymerization, centrifugation, and open-pulled (OPS) straws. Initially, Day 6 peri-hatching blastocysts were collected, cultured for 40 min in 7.5 microg/ml cytochalasin B and vitrified in 6.5 M glycerol and 6% BSA (VS1) in either heat-sealed (HS) or open straws (OS). The post-thaw survival of those stored in HS was 15.4% after 24 and 48 h in vitro; storage in OS significantly improved survival (58.8% for both 24 and 48 h). When similar stage blastocysts were cultured in cytochalasin B and vitrified with 8 M ethylene glycol and 7% polyvinylpyrrolidone (PVP; VS2) in OS, survival was 44.4 and 33.3% for 24 and 48 h, respectively. Day 5 late morulae and early blastocysts were collected, cultured with cytochalasin B, and centrifuged or left intact (control), then vitrified with VS1 in HS or OS, or vitrified in VS2 in OS only. None of the intact control embryos survived thawing and 48 h culture in vitro. Centrifuged early blastocysts vitrified with VS1 showed good post-thaw survival in culture when stored in HS (62.8 and 60.5% for 24 and 48 h, respectively), or OS (75 and 63.6%). When vitrified with VS2 in OS, survival improved (80 and 76.7%). Peri-hatching blastocysts were vitrified in VS1, and early blastocysts were vitrified with VS1 and VS2. All blastocysts were stored in OS. The embryos were recovered and transferred to Day 4 and 5 pseudopregnant recipients (for Day 5 and 6 blastocysts, respectively). Of the five recipients receiving peri-hatching blastocysts, two became pregnant and delivered a total of eight piglets. All three recipients of early blastocysts vitrified in VS1 had a delayed return to estrus; while of the four receiving embryos vitrified with VS2, two were delayed in returning to estrus, and one was confirmed pregnant after 45 days. A litter of five piglets, one male and four female, was produced at 116 days of gestation. To our knowledge, this is the first litter of piglets produced from early blastocysts vitrified without micromanipulation to remove polarized lipid droplets.

Animals↗
PubMed1993

Rat Sertoli cell extracellular matrix regulates glycosaminoglycan synthesis by peritubular myoid cells in vitro.

We have previously reported metabolic cooperation between Sertoli and peritubular myoid cells in terms of synthesis of one of the main testicular extracellular matrix (ECM) constituents, glycosaminoglycans (GAG). This study concerns Sertoli cell ECM-peritubular myoid cell interactions in terms of GAG synthesis. We have examined the responses of hormones and other regulatory agents such as a combination of follicle-stimulating hormone (FSH), insulin, retinol, and testosterone (FIRT) on peritubular myoid cells, and tested if Sertoli cell ECM or serum factor substitute for the stimulation by FIRT. Testicular peritubular myoid cells cultured on Sertoli cell ECM showed significant increases in the levels of cell- and ECM-associated GAG over that when cultured on uncoated plastic. This indicates a specific cell-substratum interaction between Sertoli cell ECM and peritubular myoid cells in the testis in terms of GAG synthesis. Moreover, in terms of cell-associated GAG synthesis, peritubular myoid cell cultured on Sertoli cell ECM or on plastic in the presence of serum substituted for the stimulatory response of FIRT on peritubular myoid cells cultured on uncoated plastic. The data are discussed in relation to the possible role of cell-substratum interaction in maintaining peritubular myoid cell functions.

Animals↗
PubMed1993

Successful fertility experiments with cryopreserved spermatozoa of barramundi, Lates calcarifer (Bloch), using dimethylsulfoxide and glycerol as cryoprotectants.

The fertility of cryopreserved Lates calcarifer sperm was studied to increase the availability of semen for routine fertilization of stripped eggs and to provide a tool for selective breeding. Semen diluted (1:4 v/v) and frozen (-196 degrees C) with 5% dimethylsulfoxide (DMSO) or 10% glycerol (final concentration) as cryoprotectants was used to inseminate freshly stripped ova. Frozen-thawed sperm were motile for about 4 min after being mixed with seawater. In the DMSO medium, post-thaw sperm activation was immediate after dilution with seawater, but in the glycerol medium maximum motility intensity was delayed for up to 1 min. When eggs and sperm were mixed before the addition of seawater, semen frozen with DMSO as cryoprotectant gave a mean hatch rate (84.1%) no different (P > 0.05) from that of unfrozen semen diluted with Ringer's solution (80.7%) or with DMSO (83.7%), but higher (P < 0.05) than that of semen frozen with glycerol (60.9%). Adding sperm to seawater 30 s before mixing with eggs did not improve the fertility of sperm cryopreserved with glycerol. Eggs inseminated with glycerol-cryoprotected sperm showed higher mortality during incubation than those inseminated with DMSO-cryoprotected sperm. Sperm held in liquid nitrogen for 90 days with DMSO as cryoprotectant yielded acceptable fertilization and hatching rates with semen-to-ova ratios of up to 1:100 (v/v) , and produced fish with no apparent abnormalities over a 29-day period after hatch. These results show that cryopreservation of L. calcarifer sperm is feasible and well suited to a variety of hatchery purposes.

Animals↗
PubMed1993

Sertoli cells as paracrine modulators of DNA synthesis in rat peritubular myoid cells in culture.

To determine whether Sertoli cells influence DNA synthesis by rat peritubular myoid cells in vitro, the effects of Sertoli cells on [3H]thymidine incorporation by peritubular myoid cells in a coculture situation were examined. Incubation of testicular peritubular myoid cells with Sertoli cells in coculture induced a significant increase in [3H]thymidine incorporation by peritubular myoid cells. This indicates a cell-cell cooperation between Sertoli and peritubular myoid cells in the testis in terms of DNA synthesis. Secreted factors from Sertoli cells, as tested in a parabiotic culture situation, also increased [3H]thymidine incorporation by peritubular myoid cells. Moreover, in terms of total cellular protein, cocultures of Sertoli cells and peritubular myoid cells resulted in a significant increase when compared with the monocultures, and this coculture effect substituted for the stimulatory response of serum on peritubular myoid cell monoculture. This study investigated the cooperative role of Sertoli cells and peritubular myoid cells in paracrine regulation of testicular functions.

Animals↗
PubMed1992

Collagen biosynthesis in cultured rat testicular Sertoli and peritubular myoid cells.

The incorporation of 3H-proline into protein was regarded as a measure of total protein synthesis and the incorporation into hydroxyproline as indicative of collagen synthesis. Relative collagen synthesis (expressed as percent of total protein synthesized) by Sertoli and peritubular myoid cells cultured from 20-22 day old rat testis was estimated. In both secreted and cellular pools, relative collagen synthesis by Sertoli cells was significantly greater than by peritubular myoid cells. Coculture of Sertoli and myoid cells resulted in a significant increase in relative collagen synthesis when compared to monocultures of each cell type. Addition of serum to peritubular myoid cells resulted in a stronger stimulation of relative collagen production. Sertoli cell extracellular matrix inhibited relative collagen synthesis by peritubular myoid cells in the presence or absence of serum. Radioactivity into hydroxyproline as corrected per cellular DNA also showed similar results. Immunolocalization studies confirmed that both cell types synthesize type I and type IV collagens. These results indicate that stimulation of collagen synthesis observed in Sertoli-myoid cell cocultures is due to humoral interactions, rather than extracellular matrix, and Sertoli cell extracellular matrix regulates serum-induced increase in collagen synthesis by peritubular myoid cells.

Analysis of Variance↗
PubMed1992

Effects of photoperiod on the reproductive physiology of male flying foxes, Pteropus poliocephalus.

Melatonion concentrations were determined in plasma pools obtained from adult male Pteropus poliocephalus (autumn mating season) at four times of the year. Melatonin levels increased within 3 h of sunset and remained elevated for the duration of the scotophase at all times of the year. Two photoperiod manipulation experiments were performed to examine the role of daylength in the regulation of the timing of the breeding season of this species. In Experiment 1, three adult males were transferred from natural short days to 16L:8D for 137 days and then photoperiod was progressively decreased over 120 days to 9L:15D; this photoperiod was then maintained for 350 days. Testicular volume (TV) peaked during decreasing photoperiod well before the time of maximal size in natural conditions. During the period in extended short photoperiod these bats showed several cycles of TV change with a progressively decreasing interval between cycles. In Experiment 2, nine adult males were exposed to the same shift to 16L:8D as in Experiment 1, but were subsequently split into three groups: 8L:16D, progressive decrease to 8L:16D and maintenance in 16L:8D. The 8L:16D and decreasing photoperiod groups showed coincident premature increases in TV, as in Experiment 1, whereas in the 16L:8D group TV increased at about the same time as in animals in natural photoperiod. Melatonin determination in the different experimental groups showed that duration of secretion was related to the length of the scotophase in all cases. These experiments demonstrate that regulation of the timing of reproduction in males of this species is influenced by changes in daylength.

Animals↗
PubMed1991

Seasonal changes in testicular size, plasma testosterone concentration and body weight in captive flying foxes (Pteropus poliocephalus and P. scapulatus).

Adult male flying foxes Pteropus poliocephalus and P. scapulatus were captured in south-east Queensland and kept in outdoor enclosures. Testicular size (TS), plasma testosterone concentrations (PTC) and body weight (BW) were measured over 1-year periods. Testicular recrudescence in P. poliocephalus began before the summer solstice and TS was greatest during mid-March (autumn) and lowest from July to September. Large increases in PTC were observed in all individuals approximately 1 month after the peak in TS. BW also increased around the time of the mating season, changes being correlated significantly with changes in TS. Mating occurred between April and June, and births from late October to late November. In P. scapulatus, TS was greatest in the spring (October) and least in the autumn (February to May); PTC fluctuated throughout the year in this species but, unlike P. poliocephalus, did not show a single large increase in the mating season. BW showed a similar seasonal pattern to that seen in P. poliocephalus, being greatest at the time of greatest TS. Mating occurred in October to November, and births in autumn. In captivity, in outdoor enclosures, these species maintained the seasonal reproductive patterns observed in the wild. The 2 species respond differently to the same environmental cues in terms of regulation of the timing of their breeding seasons.

Animals↗
PubMed1991

Mating-associated peak in plasma testosterone concentration in wild male grey-headed flying foxes (Pteropus poliocephalus).

Plasma testosterone (T) concentrations, measured in wild bats of P. poliocephalus in Queensland in 1983-87, showed a peak during the mating season in March. Plasma androstenedione (A) concentrations changed less dramatically with season. Mean testicular concentration and total content of T and A was substantially greater in March than in regressed testes in July-October. Paired adrenal glands were heavier during February to April than during September to November. In the same wild population, throughout a single breeding season (1987), plasma T concentrations were significantly higher in mid-March than 3 weeks previously or 3 weeks later. Testicular T content rose as the breeding season progressed, being greatest during March, coinciding with the large rise in plasma T concentrations. Testicular T concentration and content were correlated significantly with plasma T concentrations. Adrenal glands contained T, but the absolute concentrations were much lower than in the testis. No significant changes in plasma, testicular or adrenal A concentrations were found as the breeding season progressed. The large increase in plasma T during the mating season appears to be due to increased testicular production.

Adrenal Glands↗
PubMed1990

The cycle of the seminiferous epithelium in the Japanese quail (Coturnix coturnix japonica) and estimation of its duration.

A regular, well defined spermatogenic cycle was found in the Japanese quail by examining thin sections of isolated lengths of seminiferous tubules embedded in epoxy resin to resolve the structure of developing spermatids. The stages of the cycle initially were identified in studies using a preparatory method for fixation which separated adjacent cellular associations. The cycle was divided into 10 stages with relative frequencies (%) of Stages I to X respectively of: 11.9, 14.8, 24.1, 10.3, 8.2, 6.4, 9.4, 5.5, 3.8 and 5.4. The duration of one cycle was 2.69 +/- 0.08 days (mean +/- s.e.m.) as determined by intraventricular injection of [3H]thymidine and autoradiographic examination of the testes 1-4 days later. It was estimated that lifespans were 2.01 days for type B spermatogonia, 3.86 days for primary spermatocytes, 0.15 days for secondary spermatocytes, and 4.54 days for spermatids. The results suggest that the kinetics of spermatogenesis in the quail are fundamentally similar to the pattern in mammals.

Animals↗
PubMed1989

Limitations of salivary and blood cortisol determinations in pigs.

Blood and saliva samples were taken from groups of pigs maintained in intensive conditions. Multiple samples were taken from two unrestrained pregnant sows fitted with jugular cannulae. Single samples were taken from groups (mixed gilts and entire males; 70-90 kg) which were lightly exercised (7) or restrained (12). The rate of salivary secretion was low and collection of adequate samples took 5 min; in a number of pigs no saliva could be obtained. In order to stimulate salivary secretion, pigs (70-80 kg) were injected with pilocarpine nitrate (25 mg, subcutaneous) which produced a copious flow of saliva persisting for at least 15 min. Resting sows had higher mean levels of cortisol in plasma, ultrafiltrate and saliva than the other groups, which did not differ from each other. Within the pilocarpine group, males had a higher ultrafiltrate level of cortisol than females. In most instances salivary cortisol was significantly greater than ultrafiltrate cortisol. Ultrafiltrate and plasma cortisol were highly correlated (r = 0.883) but this correlation was low in the presence of pilocarpine (r = 0.260). Salivary cortisol was poorly correlated with either plasma (r = 0.167) or ultrafiltrate cortisol (r = 0.278) and the correlation with plasma was even lower following the administration of pilocarpine (r = 0.086). It was concluded that salivary estimates of cortisol in the pig were not usefully correlated with levels of ultrafiltrate (free) cortisol.

Animals↗
PubMed1989

Effect of exogenous estrone sulfate on embryonic survival during asynchronous transfers in the pig.

The effect of exogenous estrone sulfate (5 mg/day for 10 consecutive days starting on Day 10 after mating) on survival of embryos during asynchronous transfers was studied in Large White x Landrace gilts. Superinduction transfers were conducted by placing Day 4 embryos (younger) into mated Day-5 recipients (older) and vice versa. Treatment with estrone sulfate improved embryo survival in the transfer of younger embryos to recipients with a more developed uterine environment, but it did not affect the survival rate of older embryos in pregnant recipients. The results of the study also showed that when older embryos were transferred to a less developed uterine environment with or without estrone sulfate treatment they were better able to survine than younger embryos transferred to a more developed uterine environment.

Journal Article↗
PubMed1989

Sex steroid levels and Leydig cell ultrastructure of the male common sheath-tail bat, Taphozous georgianus.

Male sheath-tail bats were collected from central Queensland over a 12-month period. Plasma testosterone levels peaked in August, coincident with an increase in the volume of the accessory glands and ampulla/seminal vesicle secretion. Peak spermatogenesis occurred in summer and autumn and declined in the face of maximal testosterone levels in winter. Levels of androstenedione and 5 alpha-dihydrotestosterone were high compared with testosterone levels and showed no significant seasonal changes. Ultrastructural examination of Leydig cell cytoplasm revealed numerous lipid droplets and mitochondria, and an abundant smooth endoplasmic reticulum. There were no seasonal changes in Leydig cell ultrastructure. The anomalous reproductive pattern in this species is consistent with the imposition of a cold-induced winter spermatogenic shutdown, on a framework of continuous spermatogenesis, with spring peaks in testosterone and accessory gland activity.

Androgens↗
PubMed1988

Testicular migration, spermatogenesis, temperature regulation and environment of the sheath-tail bat, Taphozous georgianus.

The testes of the common sheath-tail bat of tropical Australia undergo a seasonal migration between the abdomen and the scrotal pouches, while each cauda epididymidis is permanently maintained in the scrotal pouch. Straps of smooth muscle attach to both the cranial and caudal poles of the testes, and these extend cranially to the diaphragm and caudally to the cauda epididymidis. The testicular arteries are not coiled. Among the environmental factors investigated, maximum temperature correlated most significantly with testicular descent, and the number of spermatogonia per bat also correlated most significantly with maximum temperature. Body temperature of a captive bat ranged from 25 to 38 degrees C and this was closely related to body weight and ambient temperature. It seems likely that the scrotal pouch provides a temperature slightly below that of the body and so facilitates sperm storage in the permanently scrotal cauda epididymidis. Migration of the testes probably serves to ameliorate the seasonal temperature fluctuations to which they are exposed while the relatively high correlation between maximum environment temperature and spermatogonial numbers suggests that temperature may be a proximate influence on reproduction in the sheath-tail bat.

Animals↗
PubMed1987

Prolonged epididymal sperm storage, and the temporal dissociation of testicular and accessory gland activity in the common sheath-tail bat, Taphozous georgianus, of tropical Australia.

Peak spermatogenic activity of the common sheath-tail bat occurs in autumn, declines over winter and ceases in spring. Accessory glands enlarge in spring when mating occurs, but are regressed at other times of the year. Spermatozoa are stored in the cauda epididymidis throughout the year, and their numbers increase progressively from early summer to late autumn. Sperm storage permits asynchrony of male and female cycles and allows each to be optimally timed in relation to environmental conditions. The temporal separation of primary and secondary sexual functions in the male enables the insemination of females close to ovulation and is a consequence of the burden of sperm storage being placed upon the male.

Animals↗