Biomedical subjects

G Evans

Publications and source records attributed to G Evans.

At least 19 recordsLinked to original sources

PubMed2007

Altered mRNA expression patterns in bovine blastocysts after fertilisation in vitro using flow-cytometrically sex-sorted sperm.

Sperm-sexing has been used to produce embryos and offspring of a pre-determined sex in a number of species. However, the fertility of sex-sorted sperm is reduced and the full effects of sperm-sexing remain to be elucidated. The purpose of the present study was to investigate the potential effects of sex-sorted sperm on mRNA expression patterns of developmentally important genes employing in vitro produced bovine embryos. Bovine embryos were produced in vitro with unsorted and sex-sorted sperm and mRNA expression patterns were determined for glucose-3 transporter (Glut-3), glucose-6-phosphate dehydrogenase (G6PD), X-inactive specific transcript (X-ist) and Heat shock protein 70.1 (Hsp) using semi-quantitative endpoint reverse transcriptase-PCR in male and female, day-7 and 8 embryos. The relative abundance (RA) of Glut-3 was higher for day-7 male than female embryos, and day-7 embryos derived from unsorted compared with sex-sorted sperm. The RA of G6PD was higher for embryos derived from unsorted than sex-sorted sperm, and for day-8 female compared with male embryos. The RA of Xist was higher for female than male embryos, and for day-7 female embryos derived from unsorted than sex-sorted sperm. Hsp RA was higher for female compared with male embryos, was similar for day-7 and 8 embryos, and unsorted and sex-sorted sperm derived embryos. These results demonstrate differential expression of developmentally important genes between male and female embryos, and embryos derived from unsorted and sex-sorted sperm.

Animals↗

PubMed2006

Effects of platelet-activating factor and platelet-activating factor: acetylhydrolase on in vitro post-thaw boar sperm parameters.

Cryopreservation of boar sperm compromises fertility after thawing by reducing sperm longevity and inducing acrosome reaction-like changes. In an attempt to improve the post-thaw motility and acrosome integrity of boar sperm, semen was frozen using a modified Westendorf method in which the medium was supplemented with either platelet-activating factor (PAF) or a recombinant platelet-activating factor:acetylhydrolase (PAF:AH; Pafase) before or after freezing. Platelet-activating factor is a phospholipid that is present in boar semen and PAF:AH is the naturally occurring enzyme that converts PAF to biologically inactive Lyso-PAF. Addition of PAF to the cryopreservation medium improved post-thaw motility immediately after thawing and after 3h incubation at 37 degrees C (60.0+/-0.0% and 25.0+/-2.9%; mean+/-S.E.M.) compared to the control sperm (41.7+/-1.7% and 10.0+/-2.9%; P<0.05). Acrosome integrity was higher immediately after thawing and after 3 and 6h incubation at 37 degrees C when sperm were frozen in the presence of Pafase (55.7+/-3.2%, 45.7+/-3.7% and 23.0+/-3.1%), compared to the control sperm (42.7+/-1.5%, 25.7+/-5.7% and 12.3+/-2.7%) and sperm frozen in the presence of PAF (33.0+/-3.7%, 26.3+/-2.2% and 11.7+/-0.3%; P<0.05). Addition of PAF to sperm after thawing improved motility immediately post-thaw (41.6+/-2.6%), compared with addition of Pafase (23.3+/-2.2%) or the control sperm with no supplementation of the medium (26.7+/-2.2%; P<0.05). However, this beneficial effect was lost by 3h post-thaw. Supplementation of boar semen cryopreservation medium with PAF and Pafase appeared to have beneficial effects on the in vitro quality of the sperm post-thaw.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2006

Embryo production from superovulated sheep inseminated with sex-sorted ram spermatozoa.

An experiment was undertaken to assess the fertilizing capacity of sex-sorted, frozen-thawed ram spermatozoa, artificially inseminated into superovulated ewes, and the quality and survivability of the resultant pre-sexed embryos. Synchronized (intravaginal progestagen pessary and GnRH) donors were superovulated using PMSG and repeat ovarian stimulation with FSH before insemination. Ewes (n=67) were inseminated with either 30x10(6) or 15x10(6) motile non-sorted (control) or 15x10(6) motile sex-sorted (sorted) frozen-thawed spermatozoa (control: C30 or C15; sorted: S15, respectively) and the resultant embryos transferred immediately into synchronized recipients (n=160). The percentage of transferable embryos, pregnancy rate and embryo survival were similar (P>0.05) across all treatments. Oocyte cleavage rate was higher for ewes inseminated with S15 (172/230; 74.8%; P<0.05) than for C15 (97/151; 64.2%) or C30 (89/141; 63.1%) spermatozoa. Of the lambs resulting from embryos produced with sex-sorted spermatozoa, 86/93 (92.5%) were born of the predicted sex. This study demonstrated for the first time that pre-sexed offspring derived from superovulated sheep can be produced following transfer of embryos. Furthermore, sex-sorting by flow cytometry did not compromise the in vivo fertilizing capacity of ram spermatozoa in superovulated sheep, nor did it affect the quality or survivability of the resultant embryos.

Animals↗

PubMed2006

Birth of offspring of pre-determined sex after artificial insemination of frozen-thawed, sex-sorted and re-frozen-thawed ram spermatozoa.

The fertility of ram spermatozoa cryopreserved prior to, and following, sex-sorting by flow cytometry was assessed after insemination of mature Merino ewes at a synchronised estrus. Ewes were inseminated with spermatozoa from three rams, split into four treatment groups: 50 x 10(6) motile non-sorted, frozen-thawed (Control50), 15 x 10(6) motile non-sorted, frozen-thawed (Control15), 15 x 10(6) motile sex-sorted, frozen-thawed (SF15) or 15 x 10(6) motile frozen-thawed, sex-sorted, re-frozen-thawed (FSF15) ram spermatozoa. Separation of SF15 and FSF15 treatments into X- and Y-chromosome-bearing populations was achieved using a high-speed sperm sorter. The percentage of ewes lambing after insemination was similar for Control15 (36/74; 48.6%), SF15 (35/76; 46.1%) and FSF15 (26/72; 36.1%) groups (P>0.05). A higher percentage of ewes produced lambs in the Control50 (38/70; 54.3%) than the FSF15 group (P<0.05). Fifty-one of the 55 (92.7%) lambs derived from fresh, sex-sorted frozen-thawed spermatozoa were of the predicted sex, as were 41/43 (95.3%) lambs derived from frozen-thawed, sex-sorted, re-frozen-thawed spermatozoa. This study demonstrated for the first time in any species that frozen-thawed spermatozoa, after sex-sorting and a second cryopreservation step, are capable of producing offspring of the predicted sex following artificial insemination.

Animals↗

PubMed2006

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.

Acrosome↗

PubMed2006

Non-surgical deep intra-uterine transfer of in vitro produced porcine embryos derived from sex-sorted frozen-thawed boar sperm.

Embryos and offspring of a pre-determined sex have been produced in pigs using AI and IVF with unfrozen sperm, and after surgical insemination with sex-sorted frozen-thawed sperm. The aims of this study were to demonstrate that sex-sorted frozen-thawed boar sperm could be incorporated into pig IVF for the production of embryos of a pre-determined sex and that these embryos could be successfully non-surgically transferred. Oocytes were matured in vitro, fertilised with either unsorted or sex-sorted frozen-thawed sperm and cultured until the eight-cell stage. These embryos were then transferred to recipients (n = 7) non-surgically (n = 70 embryos per sow). Oocyte cleavage was similar between sex-sorted (1538/5044; 30.5%) and unsorted (216/756; 28.6%) frozen-thawed sperm, and PCR sex-determination of the embryos confirmed that they were of the predicted sex (n = 16). Delayed return to oestrus (>23 days) was observed in five recipient sows (71.4%). Fetal sacs were observed by transcutaneous ultrasound on Day 18 in one of these sows. Pre-sexed porcine IVP embryos can be successfully produced using sex-sorted frozen-thawed boar sperm, and these embryos are capable of initiating pregnancies when transferred to recipients. However, further refinement of porcine ET protocols are required to enable development to term.

Animals↗

PubMed2006

In vitro characteristics of fresh and frozen-thawed ram spermatozoa after sex sorting and re-freezing.

The in vitro function of sex-sorted, frozen-thawed ram spermatozoa derived from fresh or frozen semen was investigated. Sorted, frozen-thawed spermatozoa had higher (P < 0.05) motility, viability, acrosome integrity and mitochondrial activity than non-sorted, frozen-thawed controls immediately following thawing and after incubation at 37 degrees C for 3 and 6 h. Similarly, frozen-thawed, sorted, re-frozen-thawed spermatozoa outperformed (P < 0.05) non-sorted controls upon thawing (mitochondrial activity) and following a 3-h incubation (motility, viability/acrosome integrity and mitochondrial activity), but there were no differences after incubation for 6 h (P > 0.05). Velocity characteristics (computer assisted sperm assessment 0-6 h post-thaw) of sorted spermatozoa derived from either fresh or frozen semen remained inferior (P < 0.05) to non-sorted spermatozoa, as did their ability to penetrate artificial cervical mucus after thawing. Direct comparison of cryopreserved spermatozoa derived from either fresh or frozen semen revealed that frozen-thawed, sorted, re-frozen-thawed spermatozoa had comparable (P > 0.05) motility, viability/acrosome integrity, mitochondrial activity, average path velocity and oviducal binding capacity immediately post-thaw, but reduced (P < 0.05) quality after 3 and 6 h of incubation. These findings indicate that, under the tested in vitro conditions, sex-sorted spermatozoa derived from fresh semen are superior in some respects to those derived from frozen semen. Further, that the use of either technique, while reducing velocity characteristics and cervical mucus penetration, results in comparable, if not enhanced motility, membrane and mitochondrial function in the post-thaw population of spermatozoa when compared with non-sorted, frozen-thawed controls.

Acrosome↗

PubMed2006

Studies on the effect of supplementing boar semen cryopreservation media with different avian egg yolk types on in vitro post-thaw sperm quality.

Fertility after insemination of cryopreserved boar semen is currently below that of fresh semen. In an attempt to improve the post-thaw motility and acrosome integrity of boar sperm, semen was frozen using an adapted Westendorf method in which the chicken egg yolk was replaced by either duck or quail egg yolk. The different composition of the yolk types, particularly the amount of cholesterol, fatty acids and phospholipids, were thought to potentially afford a greater level of protection to sperm against damage during freezing and thawing. Sperm frozen in medium containing chicken egg yolk displayed higher motility immediately after thawing, but there was no difference in the motility of sperm frozen with different types of egg yolk 3 or 6 h after thawing and maintenance at 37 degrees C. Sperm frozen in media containing chicken or duck egg yolk had a higher proportion of intact acrosomes immediately after thawing than sperm frozen in medium containing quail egg yolk, but 6 h after thawing and maintenance at 37 degrees C the sperm that had been frozen in medium containing chicken egg yolk had a higher proportion of intact acrosomes than the sperm frozen in media containing duck or quail egg yolk. Analysis of the composition of the different yolk types showed that the basic components of the yolks were similar, but the ratios of fatty acids and phospholipid classes differed. Duck egg yolk had more monounsaturated fatty acids (MUFA) than chicken egg yolk, which had more MUFA than quail egg yolk. Duck egg yolk contained more phosphotidylinositol (PI) than chicken or quail egg yolks and quail egg yolk contained more phosphotidylserine than either chicken or duck egg yolks. The differences in post-thaw motility and acrosome integrity of boar sperm when frozen in media containing the different types of egg yolk may be due to the variation in composition.

Acrosome↗

PubMed2006

The origin of membrane vesicles in ram seminal plasma.

The hypothesis tested in this study was that the membrane vesicles present in ram seminal plasma are of testicular origin, rather than being secreted by the accessory sex glands as has been previously reported for a number of species. Membrane vesicles were present in cellular extracts from reproductive organs and accessory sex glands of six rams, and in the seminal plasma of a further eight rams. When four of the latter rams were subjected to vasectomy, to isolate ejaculate contents to only the secretions of the accessory sex glands, the vesicles were largely eliminated from their ejaculates, while vesicles were still present in the ejaculates of the four control rams. The constituents of the cytoplasmic droplets and membrane vesicles derived from the seminal plasma were compared by transmission electron microscopy (TEM). Vesicles present in the cytoplasmic droplets were similar in morphology but smaller on average than those in the seminal plasma. It was concluded that the membrane vesicles in ram seminal plasma originate from either the cytoplasmic droplets, or a combination of vesicles from the droplets and the epididymis.

Animals↗

PubMed2005

Biomarkers of drug-induced vascular injury.

In pre-clinical safety studies, drug-induced vascular injury is an issue of concern because there are no obvious diagnostic markers for pre-clinical or clinical monitoring and there is an intellectual gap in our understanding of the pathogenesis of this lesion. While vasodilatation and increased shear stress appear to play a role, the exact mechanism(s) of injury to the primary targets, smooth muscle and endothelial cells are unknown. However, evaluation of novel markers for potential clinical monitoring with a mechanistic underpinning would add value in risk assessment and management. This mini review focuses on the progress to identify diagnostic markers of drug-induced vascular injury. Von Willebrand factor (vWF), released upon perturbation of endothelial cells, is transiently increased in plasma prior to morphological evidence of damage in dogs or rats treated with vascular toxicants. Therefore, vWF might be a predictive biomarker of vascular injury. However, vWF is not an appropriate biomarker of lesion progression or severity since levels return to baseline values when there is morphological evidence of injury. A potential mechanistically linked biomarker of vascular injury is caveolin-1. Expression of this protein, localized primarily to smooth muscle and endothelial cells, decreases with the onset of vascular damage. Since vascular injury involves multiple mediators and cell types, evaluation of a panel rather than a single biomarker may be more useful in monitoring early and severe progressive vascular injury.

Animals↗

PubMed2005

Post-awakening cortisol secretion during basic military training.

Salivary free cortisol concentrations in the first 30 min after awakening were assessed in 12 healthy army recruits at the beginning, middle and end of an 11-week intensive physical training course. To ensure strict adherence to protocol saliva collection was supervised and collected on each sampling day immediately on waking and again 15 and 30 min later. Self-rated psychological assessments of state levels of stress, arousal and fatigue were performed in the evening of each sampling day. A within-subjects repeated-measures analysis of participants who completed the course (12 of the original 20) showed a significant main effect of cortisol concentration across all three sampling points after awakening (F((2,22))=54.516, p<0.0001) and a significant main effect of weeks into the training course (F((3,33))=4.390, p=0.010). Further analysis of this effect of measurement-week revealed that at weeks 3 and 6 total cortisol secretion estimated by area under the curve was lower (F((3,33))=4.602, p=0.008) compared to the beginning and end of the course. Surprisingly self-reported stress, arousal and fatigue did not differ significantly across weeks, despite the large dropout rate (40%) and self-evident pressures of the course. We conclude that when controlling for many confounding variables, including participant adherence, post-awakening cortisol levels are sensitive to stressful challenge over a period of weeks.

Adolescent↗

PubMed2005

Flow cytometric sorting of fresh and frozen-thawed spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla).

We adapted flow cytometry technology for high-purity sorting of X chromosome-bearing spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla). Our objectives were to develop methodologies for liquid storage of semen prior to sorting, sorting of liquid-stored and frozen-thawed spermatozoa, and assessment of sorting accuracy. In study 1, the in vitro sperm characteristics of gorilla ejaculates from one male were unchanged (P > 0.05) after 8 hr of liquid storage at 15 degrees C in a non-egg yolk diluent (HEPES-buffered modified Tyrode's medium). In study 2, we examined the efficacy of sorting fresh and frozen-thawed spermatozoa using human spermatozoa as a model for gorilla spermatozoa. Ejaculates from one male were split into fresh and frozen aliquots. X-enriched samples derived from both fresh and frozen-thawed human semen were of high purity, as determined by fluorescence in situ hybridization (FISH; 90.7%+/-2.3%, overall), and contained a high proportion of morphologically normal spermatozoa (86.0%+/-1.0%, overall). In study 3, we processed liquid-stored semen from two gorillas for sorting using a modification of methods for human spermatozoa. The sort rate for enrichment of X-bearing spermatozoa was 7.3+/-2.5 spermatozoa per second. The X-enriched samples were of high purity (single-sperm PCR: 83.7%) and normal morphology (79.0%+/-3.9%). In study 4 we examined frozen-thawed gorilla semen, and the sort rate (8.3+/-2.9 X-bearing sperm/sec), purity (89.7%), and normal morphology (81.4%+/-3.4%) were comparable to those of liquid-stored semen. Depending on the male and the type of sample used (fresh or frozen-thawed), 0.8-2.2% of gorilla spermatozoa in the processed ejaculate were present in the X-enriched sample. These results demonstrate that fresh or frozen-thawed gorilla spermatozoa can be flow cytometrically sorted into samples enriched for X-bearing spermatozoa.

Analysis of Variance↗

PubMed2005

An efficient method of ovarian stimulation and in vitro embryo production from prepubertal lambs.

The production of embryos from prepubertal lambs is inefficient, partly resulting from the low developmental competence of prepubertal lamb oocytes, and partly because a high proportion of lambs fail to respond to hormone stimulation. The development of a hormone stimulation regimen that all lambs respond to would increase the efficiency of breeding from prepubertal animals. Using a hormone stimulation regimen consisting of oestradiol benzoate (50 microg), a norgestomet implant (1.5 mg), pregnant mare serum gonadotrophin (400 IU) and follicle stimulating hormone (130 mg) all lambs (n = 19) responded to hormone stimulation. Uterine and ovarian weight ranged from 2.8 to 7.2 g (11.8 +/- 0.7 g) and from 1.7 to 54.1 (12.5 +/- 2.9 g), respectively. The number of ovarian follicles and oocytes recovered ranged from 20.0 to 500.0 (118.2 +/- 29.2) and from 13.0 to 455.0 (82.0 +/- 24.2), respectively, and oocytes suitable for in vitro production were obtained from all 19 lambs. Uterine weight was related to both bodyweight and growth rate (P < 0.05), although ovarian weight and the number of ovarian follicles were not related to either bodyweight or growth rate. Oocyte cleavage varied between hormone-stimulated lambs (0.0-93.0%; P < 0.05), and 484/775 (62.2%) of the oocytes cultured cleaved. Oocytes from 17 of the 19 lambs (89.5%) developed to the blastocyst stage in vitro , and the proportion of zygotes forming a blastocyst (by Day 7) ranged from 0.0 to 66.7% for individual lambs. Overall, 33.9% of zygotes (n = 164) developed to the blastocyst stage, producing 8.6 +/- 2.8 blastocysts per lamb.

Animals↗

PubMed2005

Repeat ovum pick-up and in vitro embryo production from adult ewes with and without FSH treatment.

Ovum pick-up (OPU) was performed three times on adult ewes after synchronization with (n = 4) or without (n = 4) FSH treatment to investigate the effects of FSH treatment on the number of ovarian follicles, oocytes recovered, oocyte quality and development in vitro. FSH treatment increased the number of ovarian follicles (85 vs 162) and oocytes recovered (33 vs 91), although recovery rate was similar for ewes with and without FSH (91/162, 56.2% and 33/85, 38.8% respectively). Of the oocytes recovered, those classified as grades I and II were similar between ewes with (78/91, 85.7%) and without FSH treatment (27/33, 81.8%). The number of ovarian follicles was similar after repeated OPU for ewes treated with FSH, but for ewes not treated with FSH the number of ovarian follicles decreased with repeated OPU. The number of oocytes recovered decreased for FSH-treated ewes only, while the oocyte recovery rate and proportion of oocytes classified as grades I and II were not affected by repeated OPU. Oocyte cleavage (46/78, 58.9% and 19/24, 79.2%) and blastocyst formation (35/46, 76.1% and 12/19, 63.2% respectively) were similar for ewes with and without FSH treatment. The number of ovarian follicles varied between ewes (p < 0.05) although the number of oocytes recovered and oocyte development in vitro were similar between ewes.

Animals↗

PubMed2005

Current status and future directions for diagnostic markers of drug-induced vascular injury.

Recently, there has been an increased incidence of vascular toxicity in pre-clinical toxicology studies. This is of concern because of the uncertain relevance and extrapolation of this finding to humans. In dogs, profound heart rate (HR) and mean arterial pressure (MAP) changes were considered surrogate markers for drug-induced vascular injury until the early 1990s when endothelin receptor antagonists (ETRA) did not significantly alter HR or MAP but induced identical lesions in the coronary arteries of dogs. Thus significant alterations in HR and MAP were found not to be a prerequisite for this lesion. Clinically, the potential for vascular injury coupled with the lack of an unequivocal non-invasive diagnostic marker is an issue of concern to pharmaceutical companies and the regulatory authorities. Therefore, qualification and validation of biomarkers as diagnostic tools for drug-induced vascular injury would add great value to risk management and expedite the drug development process. This review focuses on the status, progress and future trends in vascular biology aimed at identification and development of diagnostic markers that are specific, sensitive and possess potential utility in both a pre-clinical and clinical setting.

Acute-Phase Reaction↗

PubMed2004

Assessment of in vitro sperm characteristics after flow cytometric sorting of frozen-thawed bull spermatozoa.

The effect of processing prior to sex-sorting, re-freezing and thawing of frozen-thawed bull spermatozoa on in vitro sperm characteristics was investigated. Frozen-thawed bull spermatozoa (three bulls; three ejaculates per bull) were prepared for sorting by washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility and forward progressive motility (FPM) after processing, staining, sorting and incubation (3 h; 37 degrees C). After frozen-thawed samples were processed and analyzed using a high-speed cell sorter, aliquots were removed and re-frozen and thawed (FTF-WASH; FTF-GRADIENT). Non-sorted frozen-thawed spermatozoa (FT-CONTROL) were also re-frozen and thawed (FTF-CONTROL). Spermatozoa from all treatments were assessed for penetration of an artificial cervical mucus at 0 h after sorting or thawing, and for motility, FPM and acrosomal status after 3-h incubation (37 degrees C). Frozen-thawed spermatozoa prepared by gradient centrifugation before sorting were sorted more efficiently than washed samples (P < 0.05). However, after sorting (FT) or thawing (FTF) and incubation, the percentage of motile spermatozoa and FPM rating was lower for GRADIENT than WASH (21.5 +/- 3.39%; 1.4 +/- 0.16 FPM versus 48.6 +/- 4.02%, 2.6 +/- 0.16 FPM; P < 0.01). Frozen-thawed sorted spermatozoa (FT) penetrated in greater numbers (151.0 +/- 19.50 spermatozoa) and distance (56.3 +/- 5.11 mm) in the artificial cervical mucus and had a higher proportion of motile spermatozoa (65.5 +/- 2.77%) and FPM rating (2.8 +/- 0.12) after incubation than spermatozoa that had been re-frozen and thawed after sorting (FTF: 14.0 +/- 3.67 spermatozoa, 21.6 +/- 3.05 mm, 12.2 +/- 1.31% and 1.2 +/- 0.10 FPM, respectively; P < 0.001). Regardless of processing prior to sorting, frozen-thawed sorted and non-sorted spermatozoa migrated similar distances in the artificial cervical mucus (FT-WASH: 60.0 +/- 1.2 mm; FT-GRADIENT: 57.2 +/- 0.76 mm; FT-CONTROL: 51.7 +/- 0.69 mm). The results of this preliminary study suggested that frozen-thawed bull spermatozoa can be efficiently sorted into high purity X- and Y-chromosome enriched samples with retained functional capacity.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

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.

Acrosome↗