In this review the concept that the origins of embryonic failure occur during oocyte development is explored. The four factors that determine oocyte viability, namely a normal growth phase, adequate follicle cell support during maturation, the completion of intracellular reprogramming before fertilization and the functioning of oocyte surveillance mechanisms, form the four sections of this review. The viability of pig oocytes at the end of the growth phase is compromised by presumptive spontaneous meiotic progression and by morphological heterogeneity. Determining the percentage and identity of viable dictyate oocytes, and identifying the reasons for the loss of viability, are key areas of future investigation. Although the requirement for follicle cell support during maturation is already established, little is yet known about the underlying signals and their transmission to the oocyte. The analysis of the action and nature of somatic signals will provide the foundation for further advances in the maturation of oocytes in vitro. Signalling cascades in oocytes control both the translation of masked mRNA and the modification and spatial localization of resultant proteins. The interdependent nature of this control system explains why inappropriate signals during maturation lead to subsequent embryonic mortality. Chromosomal errors during meiosis and early mitosis accumulate because of the leaky nature of the checkpoint system during the maternally regulated part of development: effective cell cycle surveillance is established only after the activation of the embryonic genome. In summary, we emphasize that the quality of the dictyate oocyte and the provision of appropriate signals in vitro are the principal determinants of maturational success.
The resting pool of primordial follicles in mammalian ovaries is a potential resource for the genetic manipulation of domestic animals, the preservation of endangered species, and the amelioration of some forms of infertility in humans. Exploitation of this large reservoir of follicles depends on the development of methods for activating primordial follicles to begin growth in vitro and of methods for sustaining follicular growth to the stage at which oocytes are capable of meiotic maturation, fertilization and development to live young. It has been shown that primordial follicles of rodents, cattle and primates can initiate growth in vitro, even in serum-free medium. The signals that cause primordial follicles to leave the resting pool or remain quiescent are unknown. However, of interest is the observation that in cultures of whole rodent ovaries an apparently normal number of follicles leaves the resting pool and begins to grow, whereas in cultures of isolated bovine or primate ovarian cortex almost all primordial follicles activate and develop into primary follicles. This finding suggests that non-cortical portions of the ovary may regulate the flow of follicles from the resting reservoir. In cattle, it has been difficult to sustain follicular growth beyond the primary stage and the development of methods for doing so are critical for achievement of the practical goal of use of the primordial pool for embryo production. However, the development of murine follicles in vitro from the primordial stage through oocyte maturation and fertilization, and the birth of one pup, provides encouragement for efforts to achieve similar results in large mammals.
The aims of this investigation were to quantify the changes in canine spermatogenesis that occur during ageing and to study the prevalence of testicular tumours and their effects on spermatogenesis in dogs. Testes from 74 dogs of various breeds without clinically detected testicular disease and from 28 dogs with clinically palpable tumours were examined. Testicular tumours were classified histologically according to the criteria of Nielsen and Kennedy (1990). Spermatogenesis was evaluated using a modified Johnsen score adapted for use in dogs. The diameter of the seminiferous tubules was measured in dogs without testicular disease to examine the possible effects of ageing. The different lifespans of small and large breeds were compensated for by expression as a percentage of the age at which dogs with various body weights are considered to be geriatric. Of the dogs without clinically detected disease, 21 of 74 had small testicular tumours. As in the 28 dogs with clinically detected tumours, multiple types of tumour and bilateral occurrence of tumours were common findings. The prevalence of tumours increased during ageing. Eighty-six per cent of the clinically detected tumours and 57% of the non-clinically detected tumours were found in geriatric dogs. The diameter of the seminiferous tubules did not change with age. Impairment of spermatogenesis was found only in dogs with bilateral tumours and in the affected testis of dogs with clinically detected tumours. In conclusion, it appears that spermatogenesis per se does not decrease during ageing in dogs. However, the occurrence of testicular tumours increases with age and this may affect spermatogenesis significantly.
Spermatogenesis was examined in testes from 74 dogs of various breeds without clinically detected testicular disease. A modified Johnsen score system was used to determine whether spermatogenesis deteriorates with ageing. The diameter of seminiferous tubules was measured in dogs without testicular disease to examine other possible effects of ageing on tubular performance. There appeared to be no relation between age and these variables. The influence of testicular tumours on spermatogenesis was also investigated in both affected and unaffected testes. The testes of 28 dogs with clinically palpable tumours and 21 dogs with clinically non-palpable tumours were investigated. In cases of unilateral occurrence of a tumour, impairment of spermatogenesis was observed only in the affected testis of dogs with clinically detected tumours. Bilateral occurrence of tumours, whether detected clinically or non-clinically, was associated with severe impairment of spermatogenesis. The prevalence of tumours increased during ageing. Eighty-six per cent of the clinically detected and 57% of the non-clinically detected tumours were found in old dogs. Multiple types of tumour and bilateral occurrence were very common. Seminomas and Leydig cell tumours were more frequent than Sertoli cell tumours. It was concluded that spermatogenesis per se did not decrease during ageing in dogs but the occurrence of testicular tumours increased with ageing and affected spermatogenesis significantly, as reflected by a lower Johnsen score.
Pigs begin behavioural preparations for birth about 1-2 days before parturition. Prepartum sows wander to select a suitable site and then construct a maternal nest. The signal that initiates this behavioural cascade probably results from fetal maturation but is unknown. However, endogenous PGF2 alpha appears to be involved early on in an endocrine pathway that projects to the brain and can generate most of the prepartum behavioural components. This period of intense activity is followed by a quiescent phase of lying in the nest for some hours before fetal ejection occurs. Feedback from a completed nest or abdominal discomfort may both contribute to the end of nest building. In the postpartum phase, sows have to deal with the apparently conflicting drives of remaining passive to reduce accidental or deliberate damage to piglets, while at the same time responding actively to their needs. In commercial environments, animals frequently fail in this task. Although environmental influences on piglet survival have received much experimental attention, the genetic, social and endocrine drives that control sow behaviour after parturition remain poorly understood and their clarification is a major challenge for the future.
Sperm preservation has become a routine procedure in dog breeding. In this study, the influence of prostatic fluid on sperm characteristics after preservation (either chilling or freezing) was investigated. The sperm-rich fractions of 20 ejaculates from five dogs were either extended without centrifugation or centrifuged and resuspended either directly in extender or in prostatic fluid before dilution with extender. Aliquots were processed for storage at 4 degreesC for 6h or for freezing. Storage at 4 degreesC did not affect sperm motility, viability or acrosome integrity, irrespective of the dilution treatment. However, sperm motility and viability decreased significantly after freezing and thawing, particularly in the samples with additional prostatic fluid. In contrast, the acrosome morphology of viable spermatozoa was not affected by either the dilution method or by chilling or freezing and thawing. It is concluded that addition of prostatic fluid during semen processing adversely affects the motility and viability of frozen-thawed spermatozoa. However, prostatic fluid does not appear to affect the motility and viability of chilled spermatozoa or to alter acrosome integrity in either system of preservation.
There are many potential applications of transgenic methodologies for developing new and improved strains of livestock. One practical application of transgenic technology in pig production is to improve milk production or composition. The first week after parturition is the period of greatest loss for pig producers, with highest morbidity and mortality attributed to malnutrition and scours. Despite the benefits to be gained by improving lactation performance, little progress has been made in this area through genetic selection or nutrition. Transgenic technology provides an important tool for addressing the problem of low milk production and its detrimental impact on pig production. Transgenic pigs over-expressing the milk protein bovine alpha-lactalbumin were developed. alpha-Lactalbumin was selected for its role in lactose synthesis and regulation of milk volume. Sows hemizygous for the transgene produced as much as 0.9 g bovine alpha-lactalbumin l-1 pig milk. The outcomes assessed were milk composition, milk yield and piglet growth. First parity alpha-lactalbumin gilts had higher milk lactose content in early lactation and 20-50% greater milk yield on days 3-9 of lactation than did non-transgenic gilts. Weight gain of piglets suckling alpha-lactalbumin gilts was greater (days 7-21 after parturition) than that of control piglets. Thus, transgenic over-expression of milk proteins may provide a means for improving the lactation performance of pigs.
Seven bitches in early pregnancy (12.8 +/- 3.8 days after ovulation; group 1) and seven bitches in mid-pregnancy (32.0 +/- 1.53 days after ovulation; group 2) were used in this study. For each group, five bitches were treated with 0.10 mg aglepristone (Alizine) kg-1 and this dose was repeated 24 h later. Two control bitches received a placebo. Blood samples were collected at 6 h intervals to determine plasma concentrations of progesterone, dihydro-keto prostaglandin F2 alpha (PGFM), oxytocin, prolactin and cortisol. Parturition occurred in the four control bitches. All bitches treated with aglepristone aborted. In group 1, embryonic death occurred; in group 2, fetal expulsion occurred 60-132 h after administration of aglepristone. After pregnancy termination, the interoestrous interval of aglepristone-treated bitches was significantly shorter than that before treatment. Treatment with aglepristone did not modify plasma concentrations of progesterone, prostaglandin, oxytocin or cortisol within 24 h after its administration, but it induced, in mid-pregnancy (group 2) a discharge of prolactin within 12 h after its administration. As an abortifacient, aglepristone acted on the uterus and, therefore, did not have direct or immediate luteolytic properties. Termination of pregnancy occurred with high plasma progesterone concentrations. Fetal expulsion was characterized by an increase in the concentration of PGFM, but oxytocin and cortisol remained at basal concentrations.
Although mouse embryonic stem cells have been used widely for over a decade as an important tool for introducing precise genetic modification into the genome, demonstrating the great value of this technology in a range of biomedical applications, similar technology does not exist for domestic animals. However, the development of somatic cell nuclear transfer has bypassed the need for embryonic stem cells from livestock. The production of offspring from differentiated cell nuclei provides information and opportunities in a number of areas including cellular differentiation, early development and ageing. However, the primary significance of cloning is probably in the opportunities that this technology brings to genetic manipulation. Potential applications of gene targeting in livestock species are described with particular emphasis on the generation of pigs that can be used for xenotransplantation, and the production of improved models for human physiology and disease. The development of techniques for somatic cell nuclear transfer in pigs and the challenges associated with this technology are also reviewed.
In mammals, testicular differentiation is initiated by SRY (the sex-determining region of the Y chromosome) gene expression in Sertoli cell precursors, followed by upregulation of the SOX9 gene (SRY-related HMG box gene 9). Subsequently, differentiated testis produces two hormones that induce sexual differentiation of the internal and external genital tract. Knowledge of the molecular mechanisms involved in gonadal differentiation has increased greatly over the past decade. Several genes are involved in genital ridge formation in both sexes, and others act specifically in testicular or ovarian developmental pathways. As for other mammals, relatively few data are available on the first steps of ovarian differentiation in pigs. In this review, the expression profiles of most genes known to be involved in gonadal differentiation in pigs will be presented and compared with those observed in mice. The main feature of gonadal differentiation in the pig is fetal steroidogenesis,. especially cytochrome P450 aromatase gene organization and expression. Another specific feature of gonadal differentiation in pigs is the appearance of numerous cases of XX sex-reversed animals. This intersex condition occurs as early as day 50 after coitus, during embryogenesis, and appears to be triggered genetically. It leads to a wide range of phenotypes, strikingly similar to those observed in humans. Identification of the genes involved in this pathology will improve our knowledge of mammalian gonadal differentiation and may allow the eradication of this genetic disease in pigs.
The pig industry is confronted with substantial losses due to piglet mortality. With 3-8% stillbirths and generally > 10% preweaning mortality, approximately one fifth of all fetuses formed fully at the end of gestation die before weaning. Most of these losses occur in the perinatal period. Overall prenatal development (birth weight) and specific prenatal developmental and maturational processes in late gestation are predisposing factors for perinatal losses. Birth weight and variation in birth weight remain important risk factors for perinatal mortality. Genetic selection against piglet mortality will not necessarily increase birth weight but will affect body composition and proportional organ development. Many maturational processes that occur in late gestation in preparation for extrauterine life, for example specific biochemical changes in the gastrointestinal tract, are influenced by glucocorticosteroids and are, therefore, dependent on maturation of the pituitary-adrenal system. The carbohydrate metabolism of perinatal piglets is related closely to viability in the perinatal period. The prenatal deposition of carbohydrate reserves (glycogen) and prenatal effects on perinatal glucogenic capacity, glucose homeostasis, carbohydrate metabolism and thermostability are reviewed.
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.
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 midto 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.
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.
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.
In a retrospective study, from 1994 to 1998, of inseminations with frozen semen in dogs, a total of 312 bitches of 70 different breeds were inseminated with imported (n = 183) or domestic (n = 129) semen. The overall whelping rate was 70% and mean (+/- SEM) litter size was 5.3 +/- 0.2 pups. The whelping rate was higher after intrauterine insemination (71%; n = 305) than after intravaginal insemination (29%; n = 7; P < 0.05). Timing of insemination was crucial; timing classified as optimal resulted in a higher whelping rate and larger litter size (P < 0.05) than did timing classified as early, late or too late. In the too late category, none of the bitches (n = 5) whelped. For optimal timing, whelping rate and mean ( SEM) litter size were 76% (n = 252) and 5.6 +/- 0.2, for early 33% (n = 6) and 1.5 +/- 0.5, and for late 47% (n = 19) and 2.8 +/- 0.7. Two inseminations yielded a higher whelping rate (P < 0.05) and greater mean litter size (P 0.05) than that of one insemination, 77% and 5.6 +/- 0.3, and 60% and 4.6 +/- 0.3, respectively. However, the results obtained after one insemination were poorer partly because of an overrepresentation of late insemination in this group. Semen classified as of poor quality (progressive motility < 50% or percentage of abnormal spermatozoa > 20%) gave a lower whelping rate (53%) than did semen of medium (progressive motility = 50%) or good quality (progressive motility > 50% and percentage of abnormal spermatozoa < 20%), which gave whelping rates of 76 and 74%, respectively (P < 0.05). The mean litter sizes were not significantly different. Eighty-two per cent of bitches (120 of 147) inseminated twice into the uterus at a time classified as optimal with frozen semen of good or medium quality whelped. The mean (+/- SEM) litter size was 5.6 +/- 0.3 pups in this group. These results show the potential of transcervical intrauterine insemination for routine artificial insemination in dogs.
There have been few reports of artificial insemination using fresh feline semen because of difficulties in collecting semen. It has been shown previously that the number of spermatozoa required for fertilization of cats by intravaginal insemination and unilateral intrauterine horn insemination are 8.0 x 10(7) and 8.0 x 10(6), respectively. There have been no reports of intratubal insemination of cats. Therefore, a study was designed to determine the number of spermatozoa necessary to fertilize cats by intratubal insemination with fresh semen. Four male and 25 mixed breed female cats were used. Semen was collected using an artificial vagina. Insemination was performed by laparotomy 15-30 h after administration of 100 iu hCG on day 2 or 3 of spontaneous oestrus. Cats in which ovulation had not occurred at insemination received an additional 100 iu hCG after surgery. In intratubal insemination, four groups consisting of three, seven, eight and seven cats received 5.0 x 10(3), 5.0 x 10(5), 2.0 x 10(6) and 4.0 x 10(6) viable spermatozoa, respectively, into both uterine tubes at approximately 2 cm from the fimbriae tubae. The volume inseminated was 10-20 microliters. The semen volume was adjusted with egg yolk-Tris-fructose citrate. As a result of artificial insemination, none of the cats that received 5.0 x 10(3) or 5.0 x 10(5) spermatozoa were fertilized. However, fertilization was successful in two of eight animals (25.0%) that received 2.0 x 10(6) spermatozoa and in three of seven animals (42.9%) that received 4.0 x 10(6) spermatozoa. The number of kits was 1-4 (mean +/- SE: 2.4 x 0.6). These results indicate that although large numbers of spermatozoa were inseminated into the uterine tubes, the conception rate was low. The low rate of conception may have been due to problems in inducing capacitation of spermatozoa in the uterine tubes.
The application of zona pellucida proteins for contraception of wildlife and feral animals, including stray cats, has been promoted since it was demonstrated to be effective for free-roaming feral mares. Active immunization with zona pellucida proteins leads to either reversible or irreversible infertility. Therefore, knowledge of the timing and location of zona pellucida synthesis during oogenesis in cats is a key aspect of designing an immunocontraceptive for felids. Domestic cat ovaries obtained after ovariohysterectomy were used to produce a specific rabbit antibody against feline zonae pellucidae. Ultrathin sections (70 nm) of cat ovaries were treated with the anti-zona antibody followed by incubation with gold-labelled anti-rabbit IgG for ultrastructural investigation. The gold label was related to the ultrastructure of oocytes and granulosa cells. Cat follicles at different stages of development were examined. The antibody reacted very specifically with zona pellucida proteins in fully grown oocytes with a compact zona pellucida. In secondary follicles, gold labels were found on the zona pellucida and inside granulosa cells in the vicinity of oocytes. Primary follicles were labelled inside their cubic granulosa cells and on fragments of zona pellucida in the cleft between granulosa cells and the oocyte. Some primordial follicles were characterized by labelling of the granulosa cells. In conclusion, these results indicate that cat zona pellucida is produced exclusively by granulosa cells and not by oocytes. Synthesis of zona pellucida takes place at every stage of follicular development.
Continuous low-dose administration of a GnRH analogue postpones oestrus in bitches and suppresses reproductive function in dogs. A new drug delivery formulation that could enhance the practicality of this approach for the control of reproduction has been developed. The objective of the present study was to determine whether this method of delivery could, by sustained release of the GnRH analogue deslorelin, act as a reversible anti-fertility agent in domestic male and female dogs for periods exceeding 1 year. Several long-term studies were performed, which monitored reproductive function in 30 dogs and 52 bitches. Suppression of reproductive function in male dogs was dose-related. Spermatogenesis was suppressed for more than a year in 14 of 16 dogs that received doses of > 0.25 mg deslorelin kg-1. In females, postponement of oestrus for periods of up to 27 months was observed, but there was no relationship between the stage of the oestrous cycle at the start of treatment and the duration of efficacy. Treatment-induced effects on fertility were reversible in both sexes. In summary, sustained release deslorelin implants were shown to elicit reversible long-term reproductive control in male and female domestic dogs.
