G. C. W. England

Nuclear staining and culture requirements for in vitro maturation of domestic bitch oocytes

Oocyte nuclear staining and culture requirements for in vitro maturation (IVM) in the bitch have yet to be fully investigated. In the first part of this study we investigated 7 methods for labeling nuclear material (573 oocytes). The most favorable method involved fixation plus aceto-orcein staining and light microscopy. The influence of serum supplementation of the culture medium for IVM was then investigated (1292 oocytes). Culture was performed in media supplemented with no serum or with 5, 10 and 20% fetal calf serum (FCS) and 0.3 or 4% bovine serum albumin (BSA). Identifiable nuclear material was either a germinal vesicle (GV) or GV breakdown (GVBD). After 48 h in medium plus 0, 5, 10 or 20% FCS and 0.3 or 4% BSA, the percentage of oocytes matured to GVBD was 13, 9, 15, 23, 36 and 40%, and the percentage matured to metaphase I/anaphase I/metaphase II was 4, 12, 24, 14, 36 and 13%, respectively. After 96 h, maturation to GVBD was 31, 14, 21, 11, 50 and 38%, and to metaphase I/anaphase I/metaphase II it was 6, 5, 3, 19, 15 and 9%, respectively. Within the limits of this study, BSA or high concentrations of FCS appear to be optimal for bitch oocyte maturation in vitro.

The effect of oocyte size and bitch age upon oocyte nuclear maturation in vitro

In vitro maturation in the bitch has yet to be fully investigated, and perfection of the technique is essential for future gamete salvage programs in endangered canine species. For optimal success with these techniques, knowledge of the individual animal and of oocyte effects upon maturational competence would be useful. Two factors were therefore studied using an aceto-orcein staining technique, which has been shown to be effective for monitoring nuclear maturation of canine oocytes following oocyte culture in medium supplemented with bovine serum albumin (BSA). Oocytes of different sizes were cultured in vitro and their nuclear maturation monitored. It was shown that the selection of oocytes which had acquired meiotic competence through adequate intrafollicular growth was important for in vitro maturation. In vitro maturation of oocytes from bitches aged 1 to 6 yr, and from those 7 yr and older was then compared, and it was found that oocytes from young bitches had a greater potential to mature than those collected from the older animals.

The Effect of Escherichia coli Lipopolysaccharide and Tumour Necrosis Factor Alpha on Ovarian Function

Problem  Pelvic inflammatory disease and metritis are important causes of infertility in humans and domestic animals. Uterine infection with Escherichia coli in cattle is associated with reduced ovarian follicle growth and decreased estradiol secretion. We hypothesized that this effect could be mediated by the bacterial lipopolysaccharide (LPS) or cytokines such as tumour necrosis factor alpha (TNFα).

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.

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.

Comparison of the quality of frozen-thawed and cooled-rewarmed dog semen

Abstract Semen may be preserved for short-term storage either by cooling and rewarming or by freezing and thawing. The freeze-thaw process causes significant sperm damage, and it may be preferable to cool and rewarm samples, although cooled spermatozoa has a limited lifespan. Ejaculates were collected from 6 adult male beagle dogs. The second fraction was divided into 2 aliquants, one of which was diluted with a Tris-egg yolk extender, placed into 0.5-ml straws and frozen in liquid nitrogen vapor, before being stored in liquid nitrogen. The second aliquant was diluted with a nonfat dried milk-glucose extender, cooled to and stored at 5 °C. Portions of the cooled semen were removed on Days 0, 1, 2, 4, 6, 8 and 10, and the the semen quality was evaluated after rewarming. The semen quality of the frozen semen was assessed after thawing. A variety of assessments were made, including sperm motility, sperm morphology, acrosome status, hypo-osmotic swelling, and longevity at 39 °C. Comparisons within ejaculates were made between the semen quality of the frozen samples and the samples that had been cooled and stored for different time periods. Semen quality of the samples that had been cooled and stored deteriorated on a daily basis; however, in the first 2 d after collection, semen quality was always superior to that of the samples that had been frozen and thawed. The mean time taken for semen quality of samples that had been cooled and rewarmed to become equal to samples that had been frozen and thawed varied for each parameter of semen evaluation, but overall it was 118.7 ± 25.9 h. Should short-term cryopreservation of dog semen be contemplated to allow sample transportation, it is clear that using conventional methodology dilution and cooling is the most suitable technique, provided that the sample is used within approximately 4.9 d of collection. Should the required length of storage exceed this time, it would be prudent for the sample to be frozen and then thawed.

Influence of gonadotrophin supplementation on the in vitro maturation of bitch oocytes.

IN VITRO maturation and fertilisation techniques are becoming increasingly popular for the manipulation of reproductive function in domestic animals. In carnivores, there is a need for basic investigations into oocyte maturation and the interaction of male and female gametes so that these techniques can be applied to the conservation of endangered species. Currently, two wolf and three fox species are considered to be at risk according to CITES (1997): the grey wolf (Canis lupis), the red wolf (Canis rufus), the Simien fox (Canis [Simenia] simensis), the San Joaquin kit (Vulpes macrotis mutica) and the northern swift fox (Vulpes velox hebes). These in vitro techniques may provide useful information for gamete salvage programmes, and may also allow the establishment of assays that can be used to assess the functional capacity of sperm. Despite the fact that mammalian oocytes released from the follicular environment can mature spontaneously in vitro and subsequently fertilise, their developmental ability is often lower than in vivo (Leibfried and Bavister 1983, Saeki and others 1991). This indicates that the culture systems used are inadequate models of the physiological environment and may require modification by supplementation with various factors. The intrafollicular oocyte is exposed to changing concentrations ofluteinising hormone (LH) and follicle-stimulating hormone (FSH), especially during the pre-ovulatory period. Such exposure may be essential for pre-ovulatory development of the oocyte. When in vitro maturation studies are performed, oocytes are removed from the follicular environment, and therefore from any priming effect of these gonadotrophins. The in vitro maturation studies that have been performed in the domestic bitch have generally used standard culture techniques without hormonal supplementation (Mahi and Yanagamachi 1976, 1978, Shimazu and others 1992, Yamada and others 1992, 1993). In one study, however, oestradiol was added routinely, but no effect on oocyte maturation was described (Nickson and others 1993). Previous work by the present authors also showed that there was no benefit in adding oestradiol and progesterone, alone or in combination, to a culture medium for canine oocyte maturation (Hewitt and England 1997). This short communication describes an investigation into the effect of supplementing oocyte culture medium with LH and FSH, to try to establish the effect of these factors on the follicular canine oocyte in an attempt to optimise the conditions for in vitro maturation and fertilisation. Cumulus oocyte complexes (cocs) were harvested from ovarian tissue from healthy bitches aged one to seven years. Ovarian and uterine tissue and blood samples were processed for histological analysis and plasma progesterone determination, respectively, allowing the categorisation of bitches according to the stage of the oestrous cycle at the time of ovariohysterectomy (Andersen and Simpson 1973, Eckersall and Harvey 1987). The cocs were harvested in modified HEPES-199 culture medium supplemented with 0-3 per cent bovine serum albumin (BSA) and antibiotics. The cocs were graded (Hewitt and others 1998) and Grade 1 cocs were selected for further use. The cocs were cultured in groups of up to 10 with others from the same bitch in 400 St droplets of modified HEPES199 supplemented with 0-3 per cent BSA. To the medium, either: 1 [ig/ml LH; 1 jg/ml FSH; 1 [ig/ml LH + 1 [ig/inl FSH; or no hormonal supplement was added. The LH and FSH were donated by the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK, USA). From each bitch, one half of the oocytes were cultured for 48 hours and the remaining half were cultured for 96 hours. Following culture, fixing and staining were performed as previously described (Hewitt and others 1998). Oocytes were examined using light microscopy and classified according to their degree of nuclear maturation (Hewitt and others 1998). These were germinal veside breakdown (GVBD) and metaphase I/anaphase I/ metaphase II (MI/ AI/MII). A total of 238 grade 1 cocs were obtained from 10 bitches. The stage of the oestrous cycle when each sample was obtained was determined, showing that oocytes were obtained from bitches in pro-oestrus (two), oestrus (three) and metoestrus (five). A total of 205 oocytes had identifiable nuclear material following culture. The oocyte maturation data for the 48and 96-hour culture periods were assessed using three orthogonal contrasts. These were: the overall effect of LH; the overall effect of FSH; and the interaction between these two effects. The effect of each of the treatments upon resumption (oocytes undergoing GVBD) and completion (MI/Al/MII) of meiosis were analysed separately. The data were initially assessed for the 48and 96hour culture periods combined to investigate any effect of time, treatment or their interaction (Table 1). Since there was no evidence of any of these effects for oocytes undergoing GVBD, these data were considered for the two culture periods combined. In contrast, there was evidence of an effect oftime upon oocytes maturing to MI/Al/Mll, so this analysis was performed for the 48and 96hour data separately (Table 2). It was shown that GVBD occurred in each of the culture treatments. This meant that chi-squared analysis was appropriate. In contrast, no oocytes matured to MI/AI/MII in five of the eight treatment groups: the control medium after both 48 and 96 hours; the FSH treatment after 48 hours; and both LH

Factors affecting the viability of canine spermatozoa: II. Effects of seminal plasma and blood

Abstract The second fraction from the ejaculate of six male beagle dogs was incubated together with the first and third fractions and also with blood collected from the same animal. The study demonstrates that the first and third fractions of the ejaculate, along with homologous blood, may adversely affect semen quality. These effects were, however, only manifested following prolonged incubation.

An investigation of capacitation and the acrosome reaction in dog spermatozoa using a dual fluorescent staining technique.

The processes of capacitation and acrosomal exocytosis of dog spermatozoa in vitro have yet to be fully investigated. Firstly, we investigated the effectiveness of a technique for staining dog spermatozoa with the fluorescent labels Hoechst 33258 and chlortetracycline. A modified fluorescence microscopy staining method was shown to be effective for the assessment of both viability and functional status in this species. Secondly, the presence of the ionophore A23187 in culture medium was shown to promote capacitation and the acrosome reaction of dog spermatozoa. We have therefore established that this dual fluorescent staining method can be used for monitoring these events in the dog, and it may be useful in future studies of optimal in vitro culture conditions.

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.