犬繁殖调控技术研究进展

由于犬具有一些独特的生殖生理特点,使犬的繁殖调控技术的发展一直面临着一些困难。目前犬繁殖调控技术的研究远远落后于其他哺乳动物。本文综述了犬诱导发情、精液保存、人工受精、胚胎移植及克隆等犬的繁殖调控技术国内外研究进展。     

Application of electronic estrus detection technologies to reproductive management of cattle.

Artificial insemination and embryo transfer programs are dependent on efficient and accurate detection of estrus. Visual observation is accurate at detecting animals in estrus, but efficiency ranges from approximately 50 to 70%. Electronic technologies have been developed in attempts to improve estrus detection efficiency. Commercially available electronic devices for estrus detection are based on changes in physical activity (pedometers), changes in electrical resistance of reproductive tract secretions (intravaginal resistance probes) or mounting activity (mount detectors). All of the commercially available electronic estrus detection devices can improve the efficiency of estrus detection in cattle. Pedometers are most applicable to lactating dairy cattle and have greater accuracy and efficiency when combined with visual observation. Intravaginal resistance measurement is perhaps the least practical method of estrus detection because of labor and animal handling requirements. Individual resistance measurement may have practical application for confirming other inconclusive signs of estrus. Mount monitors have the broadest application to beef and dairy cattle. HeatWatch, the only real-time radiotelometric system available, requires the least labor and animal handling and provides data on the time and duration of each mount. The less expensive stand-alone mount monitors also provide the necessary information for optimum timing of insemination and embryo transfer, but are more labor intensive.     

In vivo canine oocyte maturation, fertilization and early embryogenesis: a review

This review reports existing and original data concerning the biology of the canine oocyte and early embryo. It describes specific aspects of intra- and extra-follicular maturation of the oocyte during the peri-ovulatory period, methods to detect ovulation, sperm survival and fertilization and timing of preimplantation embryo development.     

Harvesting sperm and artificial insemination of mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female's reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.     

Cryopreservation of canine embryos

The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer, and cryopreservation of gametes have contributed considerably to the development of biomedical sciences in addition to improving infertility treatments in humans as well as the breeding of domestic animals. However, ARTs used in canine species have strictly limited utility when compared with other mammalian species, including humans. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen to date is only available for the improved breeding and reproduction for companion and working dogs as well as guide dogs for the blind. We describe here the successful cryopreservation of embryos and subsequent embryo transfer in dogs. Canine embryos were collected from excised reproductive organs after artificial insemination and subsequently cryopreserved by a vitrification method. When the 4-cell to morula stage of cryopreserved embryos were nonsurgically transferred into the uteri of nine recipient bitches using a cystoscope, five recipients became pregnant and four of them delivered a total of seven pups. The cryopreservation of embryos in canine species will facilitate the transportation and storage of genetic materials and will aid in the elimination of vertically transmitted diseases in dogs. In addition, this technique will contribute to the improved breeding of companion and working dogs such as guide dogs, drug-detecting dogs, and quarantine dogs.     

Responses of canine oocytes to in vitro maturation and in vitro fertilization outcome

The potential benefits of assisted reproduction techniques, such as in vitro maturation (IVM) and in vitro fertilization (IVF) in canids, are linked to the protection and saving of species threatened by extinction due to worldwide habitat destruction and pollution. In both domestic and wild species, these technologies will form the basis for the next leap in reproductive performance by improving fertility rates in valuable middle-aged females, by improving pregnancy rate in infertile or sub-fertile populations and by rescuing biological material to replenish populations of endangered species. In vitro techniques are supposed to answer the reproductive questions of canids, to introduce new methods for contraception and to compete with artificial insemination (AI) as the major or predominant method of embryo production, oocyte- and embryo cryopreservation and cloning. The causes affecting in vitro meiosis of dog oocytes are likely to be diverse. Incomplete understanding of the events associated with oocyte developmental competence are imputed to species reproductive physiology, medium composition and source of ovarian oocyte population used for in vitro maturation. This review addresses some issues on the current state of in vitro maturation and in vitro fertilization of canine oocytes.     

Heterochronic expression of sexual reproductive programs during apomictic development in Tripsacum

Some angiosperms reproduce by apomixis, a natural way of cloning through seeds. Apomictic plants bypass both meiosis and egg cell fertilization, producing progeny that are genetic replicas of the mother plant. In this report, we analyze reproductive development in Tripsacum dactyloides, an apomictic relative of maize, and in experimental apomictic hybrids between maize and Tripsacum. We show that apomictic reproduction is characterized by an alteration of developmental timing of both sporogenesis and early embryo development. The absence of female meiosis in apomictic Tripsacum results from an early termination of female meiosis. Similarly, parthenogenetic development of a maternal embryo in apomicts results from precocious induction of early embryogenesis events. We also show that male meiosis in apomicts is characterized by comparable asynchronous expression of developmental stages. Apomixis thus results in an array of possible phenotypes, including wild-type sexual development. Overall, our observations suggest that apomixis in Tripsacum is a heterochronic phenotype; i.e., it relies on a deregulation of the timing of reproductive events, rather than on the alteration of a specific component of the reproductive pathway.     

Current progress on assisted reproduction in dogs and cats: in vitro embryo production

The objective of the development of assisted reproduction techniques in dogs and cats is their application to non-domestic canine and feline species, most of which are considered threatened or endangered. Among these techniques, an entirely in vitro system for embryo production is effectively an important tool for conservation of wildlife. In the last decade, progress has been made in embryo production in carnivores. It has been shown that canine oocytes can resume meiosis in vitro and that these oocytes can be fertilized and developed in vitro, although at a much lower rate than most other domestic animal oocytes. The reason lies in the dissimilarities of reproductive physiology of the dog compared to other species and the lack of precise information concerning the oviductal environment, in which oocyte maturation, fertilization and early embryonic development take place. Successful in vitro embryo production in the domestic cat has been attained with oocytes matured in vitro, and kittens were born after transfer of IVM/IVF derived embryos. On the basis of these results the in vitro fertilization of oocytes has also been applied in several non-domestic feline species. The effectiveness of such protocols in the preservation of genetic material of rare species can be improved by developing better techniques for long-term storage of gametes. In dogs and cats sperm cells have been successfully frozen and the cryopreservation of oocytes would greatly increase their availability for a range of reproductive technologies. Cryopreserved cat oocytes can be fertilized successfully and their development in vitro after fertilization is enhanced when mature oocytes are frozen. Thus refined techniques of oocyte maturation and fertilization in vitro coupled with oocyte cryopreservation could allow for an easy establishment of genetic combinations when male and female gametes in the desired combination are not simultaneously available, and the propagation of endangered carnivores would be facilitated.     

Embryo and foetal loss in beef heifers between day 14 of gestation and full term

Following insemination, reproductive failure in cattle is largely manifested as embryo mortality and is a major source of financial loss to livestock producers. Ongoing studies at this laboratory into factors affecting embryo mortality have facilitated the collection of new data on the extent and timing of embryo and foetal mortality in cattle. Oestrus was synchronised in 158 beef cross heifers and following artificial insemination, embryo and foetal survival rates were determined on days 14 and 30 after insemination and subsequently at calving. Embryo survival rates measured on days 14, 30 and at full term were similar at 68%, 76% and 71.8%, respectively (P0.05). Based on morphological examination, all the 14-day-old embryos recovered were assessed as grade 1. These results provide new information indicating that most embryo losses in heifers have occurred before day 14 after insemination.     

Successful embryo transfer in the silver fox (Vulpes vulpes)

Surgical embryo transfer in the silver fox was investigated as part of a larger project concerning the conservation of endangered canine species using modern artificial reproduction techniques with the farmed fox as a model. The animals were chosen on the basis of synchrony in natural oestrus. The timing of ovulation and artificial insemination was determined by measuring electrical resistance in the vagina. Twenty-nine embryos were flushed from eight humanely killed donor females and transferred surgically into the uteri of eight recipients. One recipient female gave birth to two male pups 47 days after the transfer of four expanded blastocysts and one embryo at the 16-cell stage derived from a donor female flushed 10 days after artificial insemination.     

On net reproductive rate and the timing of reproductive output

Abstract: Understanding the relationship between life-history patterns and population growth is central to demographic studies. Here we derive a new method for calculating the timing of reproductive output, from which the generation time and its variance can also be calculated. The method is based on the explicit computation of the net reproductive rate (R0) using a new graphical approach. Using nodding thistle, desert tortoise, creeping aven, and cat's ear as examples, we show how R0 and the timing of reproduction is calculated and interpreted, even in cases with complex life cycles. We show that the explicit R0 formula allows us to explore the effect of all reproductive pathways in the life cycle, something that cannot be done with traditional analysis of the population growth rate (lambda). Additionally, we compare a recently published method for determining population persistence conditions with the condition R0 > 1 and show how the latter is simpler and more easily interpreted biologically. Using our calculation of the timing of reproductive output, we illustrate how this demographic measure can be used to understand the effects of life-history traits on population growth and control.     

Adaptive switch from infanticide to parental care: how do beetles time their behaviour?

Abstract In species where parents may commit infanticide, temporal kin recognition can help ensure parents kill unrelated young but care for their own offspring. This is not true recognition, but rather depends on accurate timing of the arrival of young and a behavioural switch from killing to caring for offspring. Mistakes have clear fitness consequences; how do species that use temporal kin recognition ensure accurate timing? We manipulated photic cues and show that the switch from infanticide to parental care in the burying beetle Nicrophorus vespilloides depends on day‐length inputs. Extending the light period after carcass discovery influenced timing of both oviposition and the cessation of infanticide. Manipulation of the light : dark cycle after oviposition also influenced timing of the switch to parental care. The timing mechanism is therefore sensitive to photic cues and access to a carcass and is not triggered by oviposition. The behavioural switch is directly related to the timing mechanism rather than changes in reproductive physiology. Given the conserved nature and extensive homology of genetic influences on biological timing, we speculate that the molecular mechanisms regulating circadian behaviour may have been co‐opted to allow beetles to determine how much time has passed after carcass discovery even though this is over 50 h.     

Canine embryonic and fetal development: a review

The progression from a fertilized oocyte to a newborn puppy is a remarkable phenomenon that occurs in a period of approximately two months. Embryonic development encompasses the period of time at which three germ layers differentiate: ectoderm, mesoderm, and endoderm. Organ systems are formed from these germ layers, with most of the reproductive tract being derived from mesoderm. Organogenesis is complete prior to the fetal stage in canine embryos, but sexual differentiation occurs during the fetal stage. Sexual differentiation is a well-coordinated progression of events that is directed initially by the genotype of the developing embryo and fetus. Developing fetuses are inherently female and will develop as such in the absence of a Y chromosome. Male fetuses develop as the Y chromosome causes regression of the female duct system and development of the male duct system. Testicular descent in the canine begins in the fetal stage, but is not completed until after birth.     

PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo

Regulation of cell cycle duration is critical during development, yet the underlying molecular mechanisms are still poorly understood. The two-cell stage Caenorhabditis elegans embryo divides asynchronously and thus provides a powerful context in which to study regulation of cell cycle timing during development. Using genetic analysis and high-resolution imaging, we found that deoxyribonucleic acid (DNA) replication is asymmetrically regulated in the two-cell stage embryo and that the PAR-4 and PAR-1 polarity proteins dampen DNA replication dynamics specifically in the posterior blastomere, independently of regulators previously implicated in the control of cell cycle timing. Our results demonstrate that accurate control of DNA replication is crucial during C. elegans early embryonic development and further provide a novel mechanism by which PAR proteins control cell cycle progression during asynchronous cell division.     

Integrating ultrasonography within the reproductive management of the collared peccary (Tayassu tajacu)

Ultrasound imaging has been used to elucidate certain aspects of the reproductive biology of wild or endangered species. However, to our knowledge, this tool has not been used for reproductive monitoring of the collared peccary (Tayassu tajacu). In this study, real-time ultrasonography was used in 16 collared peccary females to diagnose early pregnancy status and predict gestational age. Based on the detection of an embryo, the earliest pregnancy diagnosis was made on Day 18 after mating, with the mean time needed for diagnosis being 22 days. Overall accuracies on Days 22, 26 and 28 were 56, 93, and 100%, respectively. On Days 26 and 28, all pregnancy and non-pregnancy diagnoses, respectively, were correct. The fetal measurements that best correlated with gestational age were crown-rump-length (CRL) and the length and diameter of the thorax. CRL was considered the most practical measurement because, contrary to thoracic fetometry, it could be determined when the embryo was first detected. Our findings revealed real-time ultrasound scanning to be a very accurate method for early pregnancy diagnosis and prediction of gestational age in the collared peccary.     

HABITAT DIFFERENCES IN THE TIMING OF REPRODUCTION OF THE INVASIVE ALGA SARGASSUM MUTICUM (PHAEOPHYTA,SARGASSACEAE) OVER TIDAL AND LUNAR CYCLES1

Sargassum muticum (Yendo) Fensholt is an invasive species that is firmly established on intertidal and subtidal rocky shores of Europe and the Pacific coast of North America. Local success and spread of S. muticum is thought to rely on its reproductive potential that seems dependent on exogenous factors like tidal and lunar cycles. This study is the first to compare the reproductive patterns (periodicity of egg expulsion and embryo settlement) of this invader in two different habitats: the middle and low intertidal. The combination of monthly, daily, and tidal samples at triplicate sites within each habitat showed a semilunar periodicity of egg expulsion and embryo settlement coincident with increasing tidal amplitude just before full and new moons. In both habitats, duration of each egg expulsion event was ～1 week, and embryo settlement occurred during the first daily low tide and with the incoming high tide during spring tides. However, both expulsion and settlement started 1–2 d earlier, expulsion saturation was faster, and settlement was higher in the mid‐ compared to the low intertidal. Our results suggest that the exact timing of gamete expulsion and embryo release of S. muticum responds to local factors, including tidal cues, which result in differences between mid‐ and low‐intertidal habitats.     

Reproductive synchrony in a temperate damselfish, <Emphasis Type="Italic">Chromis limbata</Emphasis>

Synchronous reproduction has been reported in many tropical damselfishes, and it appears that temperature is the main driver of synchrony at these latitudes. However, reproductive synchrony and its relation to temperature is less well understood in temperate damselfish. The endemic Azores chromis (Chromis limbata) was chosen as a study species to address this topic. First, an ethogram of the reproductive behaviour and embryo development of the Azores chromis is provided. Then, are investigated (1) the reproductive dynamics at the colonial and inter-colonial levels, (2) how these are affected by temperature, and (3) the relation between reproductive dynamics and the social organization of temperate damselfish. In the Azores chromis, reproduction was cyclic with a high periodicity of 7.2 ± 0.63 days on average. Embryo development was synchronous within colonies towards the beginning and the end of incubation, but not at intermediate stages of development. Colonies that were closer were more synchronized than those located further apart, despite contrasting temperature and depth profiles. Embryo size differences were also greater between colonies that were further apart, and embryos tended to grow larger in warmer water. A literature review revealed that reproductive synchrony was common in colonial breeding damselfish species. Although these results suggest that within the breeding season, the timing and length of reproductive cycles cannot be explained by temperature alone, the observed changes in embryo size raise concern about the effects of changing oceanic conditions on this species.     

Ovulation, fertilization and early embryonic development in the bitch (Canis familiaris)

Using circulating plasma hormone estimations, ovulation was monitored in bitches. The results obtained indicate that the timing of ovulation bears little relationship to alterations in sexual behaviour. The bitches were killed and reproductive tracts were removed at various intervals after ovulation and ova or embryos were recovered. The embryo stages were assessed visually and some were investigated histologically. Embryonic development, to early blastocyst stage, took place within the oviducts during the first 12 days after ovulation and there was a marked increase in size between the early and late blastocyst. A culture system using cells from the uterine tube supported the development of one 1-cell embryo to the morula stage.     

Surgical embryo transfer resulted in birth of live offspring in farmed blue fox

Finland blue fox (Alopex lagopus) has great reputation in pelt industry around the world for its large size and top-ranking fur quality; however, both the herd size and the average survival rate of purebred offspring are rather low in production systems in China. Surgical transfer of blue fox embryos was investigated as a means to increase the population fox and also as a possible means to conserve endangered canine species. The animals were chosen on the basis of synchrony in natural oestrus. During the reproductive season of blue fox, 59 embryos were flushed from 6 farmed donors 9-11 days after the first insemination, and 53 embryos were transferred surgically into the uteri of the 6 paired recipients with natural synchronized oestrous. Two of the recipients littered 46-49 days after embryo transfer; one gave birth to 7 pups and the other 1 pup. This report describes the first successful embryo transfer in the farmed blue fox in China.     

Assisted reproductive technology in canid species

Assisted reproductive technologies in dogs began as early as the 18th century. The first scientifically recorded artificial insemination (AI) was performed in Italy by Spallanzani and lead to the birth of three pups. Progress in the area was slow, and subsequent development included AI equipment and methods for short-term preservation of fresh, and later, for frozen semen which led to the world's first litter produced from frozen semen in 1969. Improvement of freezing methods and AI equipment from 1970 onwards has rendered AI useful as a breeding technique for dogs. In parallel, AI in foxes was developed in Scandinavia in the early 1980's; this resulted in the economically valuable crossbreeding of silver and blue foxes for the production of bluefrost pelts. Unfortunately, due to the particular physiology of the canine female, progress in other artificial breeding techniques has lagged behind. Only in the last few years have these techniques been successfully applied in basic research to study oocyte maturation, in vitro fertilization, embryo cryopreservation and embryo transfer in canids.