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.

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.

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.

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.

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.

The canine oocyte penetration assay; its use as an indicator of dog spermatozoal performance in vitro.

In vitro maturation and fertilisation has yet to be thoroughly investigated in the dog and is work that is required before gamete salvage programmes can be established in endangered canine species. Due to the differences which exist between the reproductive function of Canidae and other domestic species, in vitro requirements of both the oocyte and spermatozoa may also differ, and these remain to be established. The objective of this study was to investigate the ability of in vitro capacitated canine spermatozoa to penetrate the zona pellucida of in vitro matured canine oocytes. Two methods, one which utilised the fluorescent nuclear label Hoechst 33258 in combination with an aceto-orcein stain with light microscopy, and another using the fluorescence microscopy method alone were effective for staining both oocyte nuclear material and penetrated spermatozoal heads. These techniques only rarely allowed identification of both in the same oocyte. Using this assay, two to capacitate and acrosome react in vitro as measured by the chlortetracycline and Hoechst 33258 dual fluorescence staining method. No correlation however was found between acrosomal status of spermatozoa and spermatozoal penetration of homologous oocytes, although some relationship was observed. In addition, it was found that the effect of the stage of oocyte nuclear maturation had no effect upon spermatozoal penetration and that immature oocytes could be penetrated by spermatozoa in this species.

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.

Effect of preovulatory endocrine events upon maturation of oocytes of domestic bitches.

The effect of preovulatory endocrine changes upon oocyte maturation was investigated in a combined in vivo and in vitro study. A preliminary study was performed to investigate the effect of endocrine influences in vivo on subsequent oocyte maturation in vitro. A hemi-ovariectomy was performed in four bitches during pro-oestrus at the first detected rise of plasma progesterone (mean progesterone 2.36 +/- 0.58 ng ml-1) and a second performed during oestrus immediately before ovulation, 1-4 days later (mean progesterone 4.96 +/- 0.75 ng ml-1). The mean numbers of high quality (grade 1) oocytes with identifiable nuclear material collected per pro-oestrous ovary was 15.5 +/- 9.88, and per oestrous ovary was 31.75 +/- 23.76. All of the grade 1 oocytes harvested were cultured for 96 h, after which 37% and 19% of pro-oestrous oocytes had matured to germinal vesicle breakdown (GVBD) and metaphase I/anaphase I/metaphase II (MI/AI/MII), respectively, whereas 41% and 11% of oestrous oocytes had matured to GVBD and MI/AI/MII, respectively. There was no significant difference in maturation between the two groups of oocytes. In a second study, high quality grade 1 oocytes harvested from bitches in the late luteal or anoestrous stage of the oestrous cycle were cultured in medium supplemented with either 1 microgram oestradiol ml-1 (64 oocytes), 1 microgram progesterone ml-1 (52 oocytes), a combination of oestradiol and progesterone (50 oocytes), or no supplementation (55 oocytes). The mean numbers of high quality (grade 1) oocytes with identifiable nuclear material collected per luteal ovary was 9.71 +/- 7.96, and per anoestrous ovary was 8.33 +/- 5.61. The number of oocytes obtained per bitch was highly variable, and may have been affected by the stage of oestrous cycle. After 48 h, maturation to GVBD was 56, 33, 50 and 50%, and to MI/AI/MII was 13, 17, 20 and 4% for each treatment respectively, and after 96 h was 45, 59, 63 and 63% and 10, 0, 4 and 0%, respectively. There was no significant difference in maturation between the four groups. The hormonal environment in vivo did not affect subsequent in vitro maturation of high quality oocytes. Similarly, culture in steroid hormone supplemented medium did not improve maturation in vitro of high quality oocytes. The effects upon oocytes from specific preovulatory follicles were not studied.