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.     

Trisomy-X with estrous cycle anomalies in two female dogs

Two female dogs were presented with a history of abnormal estrous cycles and infertility, despite multiple breedings. Medical therapy to correct the cycle anomalies did not result in pregnancy. Cytogenetic analysis of blood lymphocyte cultures in each dog revealed three copies of the X chromosome in each cell, constituting a 79,XXX karyotype (trisomy-X). Both dogs were eventually ovariohysterectomised and histological evaluation revealed hypoplastic ovaries and an absence of normal follicular structures. However, partial or immature follicles were noted, which may have been sufficient to cause both females to initiate cycling. The history and clinical characteristics found in these dogs were compared to those described in three other dogs reported with trisomy-X, as well as those reported in other species. These findings highlighted the importance of cytogenetic studies in fertility evaluation and achieving a definitive diagnosis for infertility in the bitch.     

Real-time PCR assays for monitoring benzimidazole resistance-associated mutations in Ancylostoma caninum

Frequent and broad application of anthelmintic drugs for treatment of intestinal parasite infection has led to drug resistance that often renders whole populations of livestock unresponsive to treatment. Therefore, it is important to detect mutations associated with drug resistance before it becomes clinically manifest. To monitor developing drug resistance against benzimidazoles (BZ), we developed real-time PCR assays and applied them to analyse the beta-tubulin isotype-1 gene of the hookworm Ancylostoma caninum, an important parasite of dogs. Previously, we developed PCR assays to monitor codon positions 167 and 200. Here, we describe an assay which is able to detect resistance alleles in codon 198. These real-time PCR assays were subsequently applied to screen hookworm specimens recovered from dogs in Georgia. No elevated levels of polymorphisms at the investigated loci were found, suggesting that selection for resistance in the tested samples did not occur.     

Focus: Zoonotic Disease: Zoonotic Ocular Onchocercosis by Onchocerca lupi

The parasitic filarioid Onchocerca lupi causes ocular disease characterized by conjunctivitis and nodular lesions. This nematode was first described in 1967 in a wolf from Georgia, and since then cases of infection from dogs and cats with ocular onchocercosis and sporadically from humans also with subcutaneous and cervical lesions caused by O. lupi have been reported from the Middle East, Europe, and North America. Due to its zoonotic potential, this parasitic infection has gained attention in the past 20 years. Phylogenetic studies have highlighted the recent divergence of O. lupi from other Onchocerca spp. and the importance of domestication in the evolutionary history of this worm. Moreover, the finding of an O. lupi genotype associated with subclinical and mild infection in the Iberian Peninsula, raises important questions about the pathogenicity of this presently enigmatic parasite.     

Common causes of male dog infertility

A complete breeding soundness evaluation is essential for assessment of the infertile male dog. Cryptorchidism, a sex-limited autosomal recessive trait, is more common as a unilateral condition. Azoospermia is an ejaculate consisting of seminal plasma but lacking sperm; repeated semen collections in the presence of an estrual bitch will rule out inadequate experience and lack of sexual stimulation. Both carnitine and alkaline phosphatase (AP) are produced in the epididymis; seminal plasma AP concentrations>5000 U/L indicate a normal ejaculate, whereas <5000 U/L is associated with incomplete ejaculation. Benign prostatic hypertrophy (BPH), the most common age-related condition in intact male dogs, is characterized by a sanguineous urethral discharge, hematuria, or hemospermia; diagnosis is based on prostatic enlargement and confirmed by a transabdominal biopsy. Although castration is recommended, valuable breeding dogs can be given finasteride. Prostatitis is more common in older dogs with BPH. Culture of the third fraction of the ejaculate or urine obtained by cystocentesis is indicated. Bacterial prostatitis is treated with antibiotics with high lipid solubility. Some dogs with bacterial prostatitis may develop prostatic abscesses (a medical and surgical emergency). Prostatic cysts are often asymptomatic. Approximately, 5-7% of dogs with prostatic disease have prostatic neoplasia, most commonly adenocarcinoma (it occurs in both intact and castrated dogs), which often metastasizes and has a very poor prognosis. Although a specific diagnosis can be made in many cases of male dog infertility, not all causes are amenable to treatment.     

Phylogeography of Bacillus anthracis in the Country of Georgia Shows Evidence of Population Structuring and Is Dissimilar to Other Regional Genotypes

Sequence analyses and subtyping of Bacillus anthracis strains from Georgia reveal a single distinct lineage (Aust94) that is ecologically established. Phylogeographic analysis and comparisons to a global collection reveals a clade that is mostly restricted to Georgia. Within this clade, many groups are found around the country, however at least one subclade is only found in the eastern part. This pattern suggests that dispersal into and out of Georgia has been rare and despite historical dispersion within the country, for at least for one lineage, current spread is limited.     

Seroprevalence of canine herpesvirus in breeding kennels in the Gauteng Province of South Africa

Canine herpesvirus (CHV-1) causes neonatal deaths as well as infertility due to embryonal death, abortion and stillbirths in breeding kennels. The aim of this study was to determine the prevalence of antibodies against canine herpesvirus in the serum of dogs older than 1 year in breeding kennels in the Gauteng Province of South Africa. A serum neutralization test (SNT) and a newly developed enzyme linked immunosorbent assay (ELISA) were used to test the serum samples of 328 dogs in 38 breeding kennels. With SNT as well as ELISA, 22% of sera were positive (P>0.9). Seventeen kennels (45% of total kennels) each had at least one positive dog on SNT compared with twenty kennels (53% of total kennels) that each had at least one positive dog on ELISA (P=0.6). The prevalence of positive dogs in positive kennels was 42+/-26% (n=17 kennels) for SNT and 39+/-26% (n=20 kennels) for ELISA. Pairwise comparison of kennels showed that the prevalence of SNT positive dogs was similar to the prevalence of ELISA positive dogs (P=0.3, n=38 kennels). Seroprevalence was independent of age, gender or colony size. This study suggests that canine herpesvirus is sufficiently common in breeding dogs in the Gauteng Province of South Africa to pose a threat to neonatal survival and fertility.     

Rabies emergence among foxes in Turkey

Sixteen rabies isolates recently collected from mainland Turkey and two isolates held within a British archive were used to form a representative cohort from a range of vectors, and were analyzed to identify potential causes for an increase of rabies within the fox (Vulpes vulpes) population in Turkey. Each isolate was characterized by sequence analysis of the nucleoprotein gene and compared phylogenetically to the cohort, to isolates from neighboring countries and to isolates from continental Europe and Russia. From this analysis the isolates could be divided into three groups associated with geographic location. This included a western group, an eastern group, and one isolate that did not group with any other Turkish isolate. This observation was also found using the heteroduplex mobility assay as an alternative method for typing rabies virus isolates. Further comparison with isolates from neighboring countries suggests that this isolate was related to viruses present in Georgia and could represent a recent import to Turkey from that country. Within the two larger groups, sequence data were obtained from both infected dogs and foxes suggesting that there has been transmission of virus between these two species. The direction of transmission could not be identified by the phylogenetic analysis, although absence of rabies within the fox population in previous years suggests that this could represent a recent spillover from the domestic dog to the fox.     

Nonsurgical fertility control for managing free-roaming dog populations: A review of products and criteria for field applications

About 75% of dogs worldwide are free to roam and reproduce, thus creating locally overabundant populations. Problems caused by roaming dogs include diseases transmitted to livestock and humans, predation on livestock, attacks on humans, road traffic accidents, and nuisance behavior. Nonsurgical fertility control is increasingly advocated as more cost-effective than surgical sterilization to manage dog populations and their impact. The aims of this review were to 1) analyze trends in numbers of scientific publications on nonsurgical fertility control for dogs; 2) illustrate the spectrum of fertility inhibitors available for dogs; 3) examine how differences between confined and free-roaming dogs might affect the choice of fertility inhibitors to be used in dog population management; and 4) provide a framework of criteria to guide decisions regarding the use of nonsurgical fertility control for dog population management. The results showed that the 117 articles published between 1982 and 2011 focussed on long-term hormonal contraceptives, such as gonadotropin-releasing hormone agonists, immunocontraceptives, and male chemical sterilants. The number of articles published biennially increased from one to five papers produced in the early 1980s to 10 to 20 in the past decade. Differences between confined dogs and free-roaming dogs include reproduction and survival as well as social expectations regarding the duration of infertility, the costs of sterilization, and the responsibilities for meeting these costs. These differences are likely to dictate which fertility inhibitors will be used for confined or free-roaming dogs. The criteria regarding the use of fertility control for dog population management, presented as a decision tree, covered social acceptance, animal welfare, effectiveness, legal compliance, feasibility, and sustainability. The review concluded that the main challenges for the future are evaluating the feasibility, effectiveness, sustainability, and effects of mass nonsurgical sterilization campaigns on dog population size and impact as well as integrating nonsurgical fertility control with disease vaccination and public education programs.     

Diagnosis and treatment of infertility in male great apes

Increased concern for maintenance of endangered species in captivity has prompted increased interest in monitoring the fertility of captive animals. This paper outlines methods for discrimination between lack of reproduction and infertility, and provides guidelines for identification of physiological causes of infertility. The role of environment in infertility is discussed, and suggestions are made for correction of environmental deficiencies. A brief review of the potential for infectious infertility is followed by an outline of appropriate treatments. It appears that a primary cause of physiological infertility in the gorilla is spermatogenic arrest, and it also appears possible that this defect may be corrected by alterations in environment and management. In summary, the outlook for continued breeding of great apes in captivity is bright, and efforts in the past few years to identify and alleviate potential causes for infertility and nonreproduction are beginning to yield good results.     

Aspects of the biology of Antarctomysis maxima (Crustacea: Mysidacea)

Summary Population structure and aspects of the reproductive biology of Antarctomysis maxima from South Georgia and the South Orkney Islands were examined. Size frequency analysis indicated that the generation time is two years at South Georgia, but may approach four years at the South Orkneys. Egg production is greater for a given size of female at South Georgia but by virtue of a larger egg, brood production in terms of ash-free dry weight is slightly greater at the South Orkneys. Young are brooded over winter at both sites and are released in spring. Females are clearly capable of breeding twice at South Georgia and it is suggested that this is also the case at the South Orkneys. These results are discussed in relation to other studies on cold water mysids and to environmental adaptations exhibited by other Antarctic marine invertebrates.     

Population structure and social organization of Southern Elephant Seals, Mirounga leonina (L.)

The population structure and social organization of the Southern Elephant Seal, Mirounga leonina , were studied at South Georgia principally by extensive field census work and determination of age and reproductive history from sections of teeth taken from samples of bulls and cows.
The adult males of the South Georgia population were exploited from 1910 to 1964, mainly at the maximum sustainable yield for this population.
The present data are compared with similar information obtained from studies at South Georgia in 1951 during the exploitation phase and at Macquarie Island in the 1950's where sealing ended in 1919 and the population had stabilized.
Changes have been noted in the time of bull haul out, number of bulls ashore, cow: bull ratio, harem size and the age of harem bulls. These changes can all be attributed to the ending of exploitation. In contrast, the structure of the cow herd has not changed appreciably in the same period.
In addition, differences in growth, body size and population structure still persist between the South Georgia and Macquarie Island populations and it is likely that most of them may reflect differences in food availability at the two locations.     

Variability in Migration Routes Influences Early Marine Survival of Juvenile Salmon Smolts

Variability in animal migratory behavior is expected to influence fitness, but few empirical examples demonstrating this relationship exist. The initial marine phase in the migration of juvenile salmon smolts has been identified as a potentially critical life history stage to overall population productivity, yet how fine-scale migration routes may influence survival are unknown. Large-scale acoustic telemetry studies have estimated survival rates of outmigrant Pacific salmon smolts through the Strait of Georgia (SOG) along the British Columbian coastline to the Pacific Ocean, but these data have not been used to identify and characterize fine-scale movements. Data collected on over 850 sockeye salmon (Oncorhynchus nerka) and steelhead (Oncorhynchus mykiss) smolts detected at an array in the Strait of Georgia in 2004–2008 and 2010–2013 were analyzed to characterize migration routes and link movements to subsequent survival at an array 250 km further along the marine migration pathway. Both species exhibited disproportionate use of the most eastern route in the Strait of Georgia (Malaspina Strait). While many smolts moved across the northern Strait of Georgia acoustic array with no indication of long-term milling or large-scale east-to-west movements, large proportions (20–40% of sockeye and 30–50% of steelhead) exhibited a different behavior, apparently moving in a westward or counterclockwise pattern. Variability in migratory behavior for both species was linked to subsequent survival through the Strait of Georgia. Survival for both species was influenced by initial east-to-west location, and sockeye were further influenced by migration timing and duration of time spent near the northern Strait of Georgia array. Westward movements result in a net transport of smolts from Malaspina Strait to the Strait of Georgia, particularly for steelhead. Counterclockwise movements may be due to the currents in this area during the time of outmigration, and the higher proportion of steelhead smolts exhibiting this counterclockwise behavior may reflect a greater exposure to wind-altered currents for the more surface-oriented steelhead. Our results provide an empirical example of how movements can affect migration survival, for which examples remain rare in movement ecology, confirming that variability in movements themselves are an important part of the migratory process.     

输卵管梗阻性不孕症的常用治疗方法及研究进展

不孕症是妇科常见疾病,其发病率呈逐年上升的趋势,在我国其发病率约为7％～10％。在女性不孕症中,输卵管梗阻是最常见的原因之一,其中又以近端阻塞多见。目前,宫腹腔镜诊断输卵管通畅度已经成为诊断输卵管梗阻胜不孕症的金标准。但是,目前治疗输卵管梗阻性不孕症的方法及效果尚未统一,常用的治疗方法不能够对患者做出全面客观的评估及准确的治疗。宫腹腔镜手术联合导丝治疗输卵管梗阻性不孕症的方法是将内镜和介入技术联合使用,较好地解决了各种治疗方法的不足,该方法凭借其可视、微创、并发症少、输卵管再通率高等优点应用于,临床。本文将对女性输卵管梗阻性不孕症的常用治疗方法及研究进展做一综述。     

Genetical population structure of Trechisibus antarcticus (Coleoptera; Car abidae) on South Georgia and on the Falkland islands

Samples of Trechisibus antarcticus (Coleoptera, Carabidae), collected on South Georgia and the Falkland Islands, were analysed at 8 polymorphic allozyme loci, related to the supposition of a recent arrival of this species on South Georgia. The data showed absence of rare alleles in the South Georgia sample, but otherwise a high similarity between the two populations.     

In focus: Georgia

Georgia is situated in the Caucasus region where Eastern Europe and Western Asia meet. The country covers a territory of 69,700 km² and its population is 4.385 million people. Formerly a part of the Soviet Union, Georgia restored its independence in 1991. Since then the country has struggled to renovate its economical and political structure, while saving the best from the past and exploring new opportunities. Bioscience is an essential part of this process. We have interviewed several leading Georgian scientists and asked them to describe the problems and accomplishments in their areas of bioscience.     

Zelkova carpinifolia (Pallas) K. Koch in Holocene sediments of Georgia—an indicator of climatic optima

Zelkova pollen has been found in Oligocene- to Pleistocene-aged deposits from many parts of Europe and northern Africa, but became extinct in mainland Europe prior to the last glacial maximum. This paper presents some observations on the ecology, pollen productivity and Holocene history of Zelkova carpinifolia to further understanding of Quaternary climatic trends. Georgia is one of the last refuges of this Tertiary relict tree.Based on palynological data from 20 Holocene sediment profiles in Georgia, we have established that Zelkova pollen is almost always accompanied by elevated proportions of thermophilous taxa (Castanea sativa, Quercus hartwissiana, Quercus iberica, and Pterocarya pterocarpa) in pollen spectra. These spectra are associated with phases of climatic amelioration and humidification. Zelkova carpinifolia is characterised by low pollen productivity and is underrepresented in pollen spectra by four-to-five orders of magnitude. Because of this, even single grains of Zelkova pollen may play a significant role in pollen-based climatic reconstructions.Six major climatic optima occurred in Georgia through the course of the Holocene, the longest and warmest of which occurred in the mid-Holocene and reached its maximum between 6000 and 5500 Cal. yr BP. During that period, Zelkova and Castanea forests were widespread. In Western Georgia, the upper tree line was elevated by as much as 300 m above its present-day level. In semiarid Eastern Georgia, the tree line may have been 500–600 m higher. Other climatic optima are seen in late-Holocene pollen spectra dating to 3800–2500 Cal. yr BP and 1350–800 Cal. yr BP.     

Y-haplotypes and idiopathic male infertility in an Indian population

Infertility being a multifactorial disorder, both genetic and environmental factors contribute to the etiology of infertile phenotype. Chromosomal anomalies and Y-microdeletion are the established genetic risk factors of male infertility. Y-haplotypes has been found as risk factor for male infertility in certain populations, though in certain others no association has been reported, suggesting a population-specific association of these variations with male infertility. In a case-control study, 165 azoo-/oligospermic patients and 200 controls were haplotyped for certain Y-haplogroups for a possible association with idiopathic male infertility in an Indian population. Analysed Y-haplogroups showed no association with infertile phenotype. Thus this genetic factor is not a risk for infertility in the studied Indian population but that does not rule out the possibility of any of them, to be a risk in other populations.     

Distribution, prevalence, and genetic characterization of Baylisascaris procyonis in selected areas of Georgia

Baylisascaris procyonis is an intestinal nematode of raccoons (Procyon lotor) that can cause fatal larval migrans in numerous species of birds and mammals, including humans. Although this parasite has historically been absent in the southeastern United States, it has been found in isolated regions in the Appalachian Mountains and was recently documented in DeKalb County, Georgia. The first objective of the current study was to investigate the distribution and prevalence of B. procyonis in selected populations of raccoons in Georgia. Intestinal tracts of 312 raccoons from 25 Georgia counties were examined for B. procyonis. The only county where B. procyonis was detected was Clarke County, where 12 of 116 (10.3%) raccoons were infected. In Clarke County, significantly more juveniles (P = 0.049) were infected compared with adults, and no differences in prevalence were noted by sex, season of capture, or land use (rural vs. urban); however, significantly (P = 0.0370) higher worm burdens were found in infected raccoons from urban/suburban locations compared with rural areas. In addition, Toxascaris leonina , a morphologically similar ascarid, was found in 3 raccoons from Clarke County (n = 2) and Morgan County (n = 1). A second objective was to determine if sequence polymorphisms were associated with B. procyonis from different geographic regions. Because sequences from a single worm from Japan had been entered into GenBank, we obtained nematodes from Kentucky and Texas for comparison with our samples from Georgia. Sequence analysis of the 18S and 5.8S rRNA genes and the internal transcribed spacer (ITS) -1 and ITS-2 regions confirmed Georgia samples were B. procyonis. Although several polymorphic bases were observed within both ITS regions, none was associated with a particular geographic location. These data indicate that the distribution of B. procyonis within Georgia is increasing and only limited genetic variation is present in the rRNA and ITS gene regions among B. procyonis from the southern United States and introduced populations in Japan.     

Preliminary results of hemizona assay (HZA) as a fertility test for canine spermatozoa

The Hemizona assay (HZA) is considered to be an effective test for predicting the fertilizing potential of spermatozoa. It is a functional test that distinguishes the zona-binding capacity of spermatozoa from fertile and infertile males. The objective of this study was to validate the HZA for canine spermatozoa, as a test for diagnosing canine male fertility status. Various parameters that affect binding capacity were examined: the presence of an adequate number of capacitated and motile spermatozoa for an HZA, the influence of fertility status, sperm-binding variability within fertile dogs over 60 d, variability in sperm-binding capacity of different oocytes, the lower limit number of spermatozoa binding to a zona from the fertile control, and evaluation of HZI to determine the fertilizing capacity of spermatozoa. Hemizonae were obtained from frozen oocytes of spayed bitches. The oocytes were manually cut into nearly equal halves. Spermatozoa were capacitated by swim-up and 1 h incubation at 37 degrees C in modified Ham's F10 medium. Spermatozoa and hemizonae were co-incubated in 100-microL drops at 37 degrees C for 1 h. Spermatozoa from 7 fertile and 3 infertile dogs were used for this study. The optimal sperm concentration for hemizona insemination was 1 x 10(6)/mL capacitated and motile spermatozoa. A significant difference (P < 0.001) was found the number of tightly zonabound spermatozoa between fertile and infertile dogs. Although there was a small difference in zona binding capacity between ejaculates of the same fertile dog (44 +/- 18.24), the main cause for the difference mentioned above was that of poor zona pellucida-binding capacity of spermatozoa from infertile dogs. We found a maximum of 14.28% bad oocytes when we compared sperm samples from 3 fertile and 3 infertile dogs in 56 HZA replicates. To avoid the effect of bad zona on sperm binding we calculated 37 (95% CI) bound spermatozoa from infertile dogs in 56 replicates. Thus, an HZA experiment in which a control dog had < 37 zona bound spermatozoa was repeated. Based on a minimum of 37 bound spermatozoa for fertile males (controls), a differential zona-binding capacity and hemizona index (HZI) between fertile and infertile dogs and between 2 fertile dogs was determined. The binding differential between fertile and infertile dogs was 64.92 +/- 24.29, while between 2 fertile dogs it was 22.38 +/- 10.02 (P < 0.001). According to the HZI values, a value equal to or less than 41.11 indicated an infertile test dog, while an HZI value equal to or greater than 57.95 indicated a fertile test dog. Any value between these two could indicate either fertility or infertility. The evaluation of fertilizing potential of spermatozoa can be improved using the HZA protocols described above.