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Biomedical subjects

G C England

Publications and source records attributed to G C England.

At least 19 recordsLinked to original sources
PubMed2001

Cryopreservation of epididymal dog sperm.

A method of cryopreservation was developed for sperm salvaged from the cauda epididymis and vas deferens of domestic dog testes. Four modifications of the glycerol concentration of a buffer used for cryopreservation of dog ejaculates and two freezing rates were assessed for their effect upon post-thaw spermatozoal motility and morphology. There was no statistical difference between the four glycerol concentrations or the two freezing rates and the buffer containing 6% glycerol and the freezing rate provided by 0.5 ml straws was chosen for further study. This method resulted in a significant reduction in the percentage of live spermatozoa detected with Hoechst staining and a reduction in the percentage of capacitated spermatozoa after freeze-thawing. However, there was no difference in the ability of frozen-thawed spermatozoa to penetrate homologous oocytes. This study demonstrates that cryopreservation of epididymal canine sperm can be performed using methods similar to those established for ejaculates of the same species, and that despite some damage, spermatozoa retain their functional ability.

Animals↗
PubMed2001

Solubilized zona pellucida proteins and progesterone induce calcium influx and the acrosome reaction in capacitated dog spermatozoa.

Spermatozoa from the sperm-rich fractions of the semen of 6 beagle dogs were capacitated and the effect of both zona pellucida (ZP) proteins and progesterone on calcium flux and the acrosome reaction measured. Sperm calcium flux was determined using the dual wavelength calcium probe indo-1/AM (6 microM) in a flow cytometric assay (one ejaculate from each dog examined; n = 6). No calcium flux was observed in the negative control treatments (RPMI medium or DMSO). Both heat-solubilized bitch ZP proteins and progesterone caused a similar response characterized by a gradual but marked influx of calcium ions which was sustained over 2 min. Acrosomal status was assessed by indirect immunofluorescence using a specific monoclonal antibody following 1 hr incubation for each treatment (four ejaculates from each dog examined; n = 24). The level of acrosomal exocytosis was very high for samples treated with ZP proteins (70.3 +/- 2.1%) and progesterone (84.6 +/- 1.5%) and was significantly different from the respective controls (P < 0.001). Interestingly the patterns of calcium flux in response to both ZP proteins and progesterone were in contrast to the situation in other species studied to date raising the possibility that the mechanism for triggering the acrosome reaction may be different in dog spermatozoa. In addition the high degree of progesterone-induced acrosomal exocytosis compared to other species raises the probability that the majority of dog spermatozoa are already undergoing the acrosome reaction before they reach the egg ZP.

Acrosome↗
PubMed2001

Effect of romifidine on gastrointestinal motility, assessed by transrectal ultrasonography.

A technique of transrectal ultrasonography was developed to investigate the effects of romifidine 80 and 120 microg/kg bwt on intestinal motility in the horse. Motility of the small intestine, caecum and left ventral colon were assessed following injection of romifidine and a saline control, using a blinded, cross-over study design in 6 horses. Measurements were taken at 15, 30, 60, 120, 180 and 240 min after drug administration. There was a slight nonsignificant decrease in motility in the control group over the 4 h study period. Both doses of romifidine produced a marked decrease in gastrointestinal motility and were associated with the presence of reduced (nonpropulsive) contractions. Transrectal ultrasonography proved suitable for monitoring changes in the type and frequency of intestinal motility in the horse.

Adrenergic alpha-Agonists↗
PubMed2001

Manipulation of canine fertility using in vitro culture techniques.

Highlighting the important aspects of canine reproductive physiology, this review documents developments made with in vitro culture of canine gametes, and indicates how these techniques can be further exploited. In vitro culture of gametes and embryos has been achieved in many mammalian species, allowing the development of reproductive technology. Despite numerous investigations in other species, only a small number have been performed in dogs. Further studies are required to establish the differences between canine reproductive physiology and that of other mammals. Ultimately, these studies may lead to the development of a gamete salvage technique incorporating oocyte, spermatozoa and embryo culture, and cryopreservation to allow genetically important material from Canidae to be saved. In dogs, the oocyte is ovulated when immature rather than mature, as in other species, and the survival rate of spermatozoa is greater within the female tract. By incorporating knowledge of canine reproductive physiology and work on in vitro culture in other mammals, some important achievements have been made. Sperm capacitation, induction of the acrosome reaction, maturation of canine oocytes and fertilization have all been achieved in vitro. Embryo culture has proven more difficult, with only two studies reporting success. Cryopreservation of spermatozoa has been perfected and is used routinely when transporting semen for artificial insemination. However, improvement in embryo culture techniques and development of oocyte and embryo freezing are required. This work in the domestic model may lead to development of a gamete-banking programme to safeguard the future of endangered canine species.

Acrosome Reaction↗
PubMed2001

Test of canine sperm function in vitro using primary homologous oocytes with fluorescence and confocal microscopy.

In vitro capacitated canine spermatozoa can interact with both mature and immature homologous oocytes in vitro, but it is difficult to determine whether spermatozoa have actually penetrated the oocyte or have simply bound to the zona pellucida. The aim of the first part of this study was to determine accurately the location of spermatozoa in relation to the oocyte by comparing observations made using confocal and fluorescence microscopy. The fluorescence technique had a sensitivity of 66%, and correctly identified 98% of the spermatozoa that had penetrated or bound oocytes and could discriminate between penetrated and bound spermatozoa with a sensitivity and specificity of 97% and 100%, respectively. This finding demonstrates that evaluation of the assay by fluorescence microscopy is reliable for detecting the presence and location of spermatozoa in relation to homologous oocytes. The aim of the second part of the study was to attempt to simplify the assay using fluorescence microscopy and immature oocytes. No significant difference was found between sperm interaction with mature and immature oocytes, which demonstrates that culturing oocytes before the assay has no benefit, and that the assay can be performed quickly and easily using non-cultured oocytes to provide rapid evidence of the fertilizing potential of spermatozoa.

Animals↗
PubMed2001

Effect of technical settings and semen handling upon motility characteristics of dog spermatozoa measured using computer-aided sperm analysis.

Technical aspects of computer-aided sperm analysis and the influence of semen preparation were investigated for their effect on the measured motility characteristics of dog spermatozoa. Altering the internal image settings significantly influenced the measured motility by changing the ability of the computer to recognize spermatozoa. Similarly, the use of a longer minimum track point (the minimum length of sperm track detected before analysis) resulted in failure to detect some of the faster moving spermatozoa. There was a clear interaction between the search radius (the threshold distance below which objects are linked together) and the minimum track point. A 1 min analysis period was required to eliminate reduced motility as a result of sample deterioration upon the microscope stage. The dilution of semen to between 1:10 and 1:20 was necessary to allow accurate detection of sperm motility; however, such dilution significantly altered the motility characteristics of spermatozoa. The influence of viscosity and ionic composition of the media was confirmed by comparing dilution in seminal plasma with dilution in iso-viscous methylcellulose and iso-osmotic saline, respectively. Analysis temperature had a significant influence on sperm motility, although values were most constant within the range of 25-45 degrees C. Extremes of temperature had marked deleterious effects. Careful selection of internal image settings, the minimal track point and search radius, and the analysis time are essential for accurate detection of sperm motility. Moreover, dilution of spermatozoa per se, and dilution with media of different viscosities and ionic compositions can alter the sperm motility. Once these aspects of computer image analysis are determined for each system, the method can achieve a high degree of repeatability with interanalysis coefficients of variation of < 12%, and intra-analysis coefficients of variation of < 3% for most parameters.

Animals↗
PubMed2001

Vital and ultrastructural changes in dog spermatozoa during cyopreservation.

The exact nature of cryo-injury to dog semen and the stage of cryopreservation at which it occurs have not been evaluated fully and were investigated in the present study. Semen samples were examined immediately before and after addition of semen extender, cooling and freeze-thawing in a Tris-based medium. Vital and ultrastructural assessments were made immediately after each treatment and after incubation for 4 h at 39 degrees C after each treatment. Addition of semen extender produced no significant ultrastructural changes in spermatozoa; however, cooling resulted in an immediate increase in the number of acrosomal abnormalities and a subsequent decrease in sperm viability. Freeze-thawing caused both an immediate and a delayed decrease in sperm viability. This effect may have been an exacerbation of the effects of cooling as many acrosomal abnormalities were present or it may have been the result of initially unrecognized damage. Cooling and freezing of dog spermatozoa may have both immediate and delayed effects on the ultrastructure of spermatozoa. The immediate effects of cooling and freezing may either kill spermatozoa or render them incapable of fertilization by damaging the acrosome, whereas delayed effects may reduce sperm longevity by altering plasma membrane structure.

Acrosome↗
PubMed2001

Cystic endometrial hyperplasia/pyometra in dogs: a review of the causes and pathogenesis.

In this review the roles of endogenous and exogenous steroid hormones, steroid receptors, spontaneously occurring and experimentally induced genital tract infection, and endometrial trauma in the cause and pathogenesis of cystic endometrial hyperplasia/pyometra in bitches are considered. It is postulated that intrauterine bacteria, which ascend from the vagina during pro-oestrus and oestrus, induce the disease during metoestrus by acting on the progesterone-primed endometrium directly via toxin production, or indirectly by the release of inflammatory mediators. The lesion can be produced by endometrial trauma in a sterile uterus without the systemic effects associated with the disease. There is no association with premature or increased progesterone concentrations at the time that the disease is normally diagnosed in mid- to late metoestrus; however, such aberrant endocrine changes cannot be excluded from involvement in the pathogenesis of this disease, as there are no data from bitches preceding the onset of clinical signs. Expression of oestrogen and progesterone receptors are modified by endogenous steroid hormones but there is no clear evidence that changes in these receptors are involved in the pathogenesis of the disease; receptor expression can be modified by exogenous hormones.

Animals↗
PubMed2000

Cardiopulmonary effects of romifidine and detomidine used as premedicants for ketamine/halothane anaesthesia in ponies.

The cardiopulmonary effects of romifidine at 80 microg/kg (R80) or 120 pg/kg (R120), and detomidine at 20 pg/kg (D20) when used as premedicants for ketamine/halothane anaesthesia were investigated in six ponies. Using a blinded crossover design, acepromazine (0-04 mg/kg) was administered followed by the alpha-2 agonist. Anaesthesia was induced with ketamine at 2.2 mg/kg and maintained with halothane (expired concentration 1.0 per cent) in oxygen for three hours. During anaesthesia, arterial blood pressure, cardiac index, PaO2 and PmvO2 decreased, and systemic vascular resistance and PaCO2 increased. The cardiac indices for R80, R120 and D20 were, respectively, 39, 39 and 32 ml/kg/minute at 30 minutes and 29, 29 and 26 ml/kg/minute at 180 minutes. The alpha-2 agonists had similar cardiovascular effects, but PaO2 was significantly lower with R120. The quality of anaesthesia was similar in all three groups.

Analgesics↗
PubMed2000

Investigation of romifidine and detomidine for the clinical sedation of horses.

The effects of two intravenous doses of romifidine (80 and 120 microg/kg) and one dose of detomidine (20 microg/kg) were compared in a blinded study in 30 horses requiring to be sedated for routine dental treatment. Several physiological parameters were assessed before and for two hours after the administration of the drugs, and the horses' teeth were rasped 30 minutes after they were administered. Romifidine produced a dose-dependent effect on most parameters. Detomidine at 20 microg/kg was similar to romifidine at 120 microg/kg in the magnitude of its sedative effects, but was similar to romifidine at 80 pg/kg in its duration. There were no significant differences between the three treatments in terms of the clinical procedure score.

Analgesics↗
PubMed2000

Contact of dog spermatozoa with homologous uterine tube epithelium prolongs flagellar activity in relation to the stage of the estrous cycle.

There is scant information about the storage of spermatozoa within the reproductive tract of the bitch. In several species the uterine tube plays a significant role in sperm storage. The present study was performed to investigate the interaction between spermatozoa and the epithelium of the uterine tube, in particular how this interaction might influence the flagellar activity of spermatozoa in relation to the stage of the estrous cycle. Epithelium was harvested from uterine tubes of 24 bitches at various stages of the estrous cycle (estrus, luteal phase or anestrus), and cultured with pooled spermatozoa collected from 6 dogs. Spermatozoa rapidly bound to the epithelial surface by their heads and the majority of attached spermatozoa were motile. The intimate association between spermatozoa and the uterine tube epithelium maintained motility in a manner that was related to the stage of the estrous cycle. Flagellar activity was significantly greater for spermatozoa bound to estrous epithelium than epithelium from the luteal phase or anestrus. On average, approximately 10% of spermatozoa that were attached to the uterine tube epithelium of estrous bitches retained their flagellar activity for 48 h after innoculation. There was no apparent influence of the region of the uterine tube on this effect. These findings suggest that the uterine tube may form a functional spermatozoal reservoir in the bitch.

Animals↗
PubMed2000

Ultrasonography of spontaneous lesions of the genital system of three rams, and their influence on semen quality.

Three young Suffolk ram lambs had lesions which rendered them reproductively unsound. One had a unilateral scrotal hernia, detected by palpation, and two had sperm granulomas which were detected by trans-scrotal ultrasonography at 18 to 20 weeks of age, before they could be detected by palpation. Changes in the lesions were monitored ultrasonographically at two-week intervals with a 7.5 MHz linear array transducer. On the day of each examination, semen was collected from the rams with sperm granulomas by using an artificial vagina, and their libido was assessed. Semen could rarely be collected from the third ram. Comparisons were made with similar data obtained from two normal rams. When the rams were 20 to 21 months of age, they were vasectomised on the normal side, so that the influence of the lesions on semen composition and libido could be assessed, and semen was collected and evaluated until they were euthanised at approximately 24 months of age, when the genital system was examined for gross lesions. The sperm granulomas changed in size (one of them increased in size by about 30 times) and echotexture over the 19-month period of the study, whereas the scrotal hernia, which contained omental fat, remained fairly constant. The libido of the two rams with sperm granulomas was comparable with the two normal controls. The total mean (sem) number of sperm in the ejaculates were 1.11 (0.10) x 10(9) and 2.03 (0.17) x 10(9) sperm/ml in the affected rams and 4.11 x 10(9) sperm/ml in the normal rams over the same period; the differences were significant (P<0.05). After unilateral vasectomy, the ejaculates from these two rams contained small numbers of dead spermatozoa, mostly with degenerate heads. Postmortem examination confirmed the gross lesions identified ultrasonographically.

Animals↗
PubMed1999

Oestrogen and progesterone receptors in the uterine wall of bitches with cystic endometrial hyperplasia/pyometra.

Fresh samples of uterine wall and peripheral plasma were obtained from 13 bitches of different breeds when they were treated for cystic endometrial hyperplasia/pyometra by ovariohysterectomy. The plasma samples were assayed for progesterone and 17beta-oestradiol, and the tissue samples were examined histopathologically and for the presence of oestrogen and progesterone receptors by an immunocytochemical method. The immunoreactivity was scored semiquantitatively, taking into account both the intensity and distribution of the specific staining of the receptors, by using a simplified scoring system. The scores for both oestrogen and progesterone receptors in the glandular epithelium were much lower, and in the endometrial stroma a mean (sd) score for oestrogen receptors was 46.0 (44.7) compared with 0 in comparable endometrial tissues from normal bitches at the same stage of the oestrous cycle.

Animals↗
PubMed1999

Semen quality in dogs and the influence of a short-interval second ejaculation.

Semen quality was examined in each of 65 known fertile dogs. Values were found to be similar to those previously published, although an apparent breed influence was demonstrated, with German shepherd dogs producing ejaculates of larger volume and greater total spermatozoal output than other breeds. A second ejaculate was collected from each dog with a mean interval of 63 min. The second samples had significantly lower values for the volume of the second fraction, the spermatozoal concentration and the total spermatozoal output. There were no differences for the percentage motility or the percentage of morphologically normal live spermatozoa. While there was no increase in semen quality of the second ejaculate, the technique may be useful since it results in the collection of approximately 70% more spermatozoa than a single ejaculate. These spermatozoa also had normal motility and morphology and could, therefore, be used for insemination or cryopreservation.

Animals↗
PubMed1999

The influence of exogenous steroid hormones on steroid receptors, uterine histological structure and the bacterial flora of the normal bitch.

Oestrogen (ER) and progesterone (PR) receptors have been shown to vary in both concentration and distribution during the oestrous cycle of the bitch, influenced by the normal changes in endogenous reproductive hormones. The influence of exogenous steroid hormones on steroid receptors and the histological structure of the uterus was studied in two groups of parous Beagle bitches. Group A (n = 6) were treated with progesterone (P4) in oil i.m. (3 mg/kg) in late metoestrus on the day that peripheral plasma P4 concentrations were first identified as <10 ng/ml, and subsequently once weekly on three other occasions. Group B (n = 6) were treated with a single i.m. injection of MPA (50 mg, 4.2-5.6 mg/kg) following the same protocol. Full-thickness uterine wall biopsies were obtained from the mid part of one horn 2-7 days after the last (fourth) injection of P4 or MPA. During the subsequent oestrus, when peripheral plasma P4 concentrations were between 8 and 10 ng/ml, each bitch in both groups (n = 12) received a single injection of oestradiol benzoate (ODB) in oil i.m. (7.5 mg, 0.63-0.84 mg/kg). All bitches had an ovariohysterectomy 7 days later. Full-thickness uterine wall samples were obtained from the mid part of the intact horn and other parts of the uterus. Swabs were taken from the uterine lumen for bacteriological examination; all were sterile. Tissue samples were sectioned and examined for evidence of lesions, and stained for ER and PR receptors using an immunocytochemical method. The immunoreactivity was scored semiquantitatively, incorporating both the intensity and distribution of specific staining of the receptors using a simplified histoscore (H-score). At the time of ovariohysterectomy, fluid had accumulated in the isolated section of the uterine horn distal to the point of biopsy; the volume was greater in the MPA-treated bitches. There was also evidence in some sections of histological changes in the endometrium. Variations in the expression of both ER and PR were seen between bitches, which may have been due to some not being in mid-metoestrus at the time of treatment. In general, ER scores were low after P4 and MPA treatment, but following ODB there was a significant (P<0.05) increase in ER expression in all parts of the endometrium. PR scores were zero in the glandular epithelium of all 12 bitches after P4, MPA and ODB treatment, whereas in the other parts of the endometrium they were generally moderate to high. Following treatment with ODB, PR generally increased in the three regions of the endometrium where PR were present. The study shows that ER and PR distribution and expression in the endometrium of bitches can be modified by P4, MPA and ODB, with evidence of individual variation.

Animals↗
PubMed1999

Synthetic oviductal fluid and oviductal cell coculture for canine oocyte maturation in vitro.

The perfection of in vitro maturation in the bitch has yet to be achieved, and is an essential prerequisite for gamete salvage programmes in endangered canine species. In contrast to most mammals, the bitch ovulates an immature oocyte which undergoes meiotic maturation within the oviduct. A model of the oviductal environment may therefore be useful for performing in vitro maturation. This study was performed to investigate the effect of introducing an oviductal element to the culture environment, first with the use of a synthetic oviductal fluid (SOF), and secondly, using coculture with isolated canine oviductal epithelial cells, upon the rate of oocyte maturation in vitro. It was found that there was no difference in the proportion of oocytes undergoing germinal vesicle breakdown (GVBD) after 48 h in culture between SOF containing 0.3% bovine serum albumin (BSA, 45%), containing 4% BSA (36%) and control medium 199 (27%). There was also no difference in oocyte nuclear maturation to metaphase I/anaphase I/metaphase II (MI/AI/MII) after 48 h in culture between SOF containing 0.3% BSA (5%), containing 4% BSA (7%) and control medium 199 (6%). In addition, there was no difference in oocyte nuclear maturation to MI/AI/MII after 96 h between SOF containing 0.3% BSA (0), containing 4% BSA (7%) and control medium 199 (11%). In contrast, the proportion of oocytes undergoing GVBD after 96 h in culture was affected by the treatment used, with 27% in SOF + 0.3% BSA, 62% in SOF + 4% BSA and 63% in medium 199. It was found that there was no difference in the proportion of oocytes undergoing GVBD between the coculture treatments 199 (33%), 199 + cells (37%), coculture medium (30%) and coculture medium + cells (49%), and for oocyte nuclear maturation to MI/AI/MII, between medium 199 (2%), 199 + cells (0), coculture medium (6%) and coculture medium + cells (2%) after 48 h in culture. In addition, there was no difference in oocyte nuclear maturation to GVBD after 96 h between 199 (61%), 199 + cells (59%), coculture medium (65%) and coculture medium + cells (53%). In contrast, the proportion of oocytes maturing to MI/AI/MII after 96 h in culture was affected by the treatment used, with a significant difference between 199 (0), 199 + cells (9%), coculture medium (0) and coculture medium + cells (0). It was shown, therefore, that the culture of oocytes in the SOF improved oocyte nuclear maturation when supplemented with a high concentration of protein and that culture in the presence of oviductal epithelial cells improved oocyte maturation, but only after a prolonged period of time.

Animals↗