Changes in interleukin-2, -4, -6 and -8 expression in the postovulatory sow endometrium after artificial insemination based on conceived or failed to conceive.

The establishment and maintenance of a pregnancy requires proper interaction between the endocrine and immune systems in the uterus. Therefore, it is crucial to understand how changes in endometrial cytokine levels facilitate reproduction. This study aimed to investigate how representative cytokines sequentially changed in the endometrium and whether conception could be attributed to these changes. In this study, artificial insemination was performed twice in 160 sows and ovulation was examined every 3 h using transrectal ultrasonography. Uterine endometrial tissues were obtained via repeated biopsies at 2, 4, 6, 8, 12, 16, and 20 h after ovulation and interleukin (IL)-2, IL-4, IL-6, and IL-8 expression was examined using real-time polymerase chain reaction. The conception rate was 91.9%. The IL-2 levels showed no differences in conception or time. The expression peaks of IL-4 and IL-6 were delayed in sows that failed to conceive within 4-6 h and 2 h, respectively, compared to those that did conceive. In sows that conceived, IL-8 was highest after 2 h, and no difference was observed at other time point, regardless of conception. In sows that failed to conceive, the increase in IL-8 levels might have been cancelled or terminated before the first sampling time. These results highlight the importance of timely increases and subsequent declines in the levels of some cytokines for the establishment of pregnancy. Differences in uterine capacity start just after ovulation; detection and correction of these deviations can improve the reproductive efficiency of sows.

A Case Report of Acute Cardiac Tamponade Creation in a Macaque: Echo-Guided Catheter Manipulation to Perforate Coronary Artery.

Although acute cardiac tamponade is one of the major problems in clinical practice, a suitable animal model is still lacking. We tried to create acute cardiac tamponade in macaques by echo-guided catheter manipulation. A 13-year-old male macaque was anesthetized, and a long sheath was inserted into the left ventricle via the left carotid artery under the guidance of transthoracic echocardiography. The sheath was then inserted into the orifice of the left coronary artery to perforate the proximal site of the left anterior descending branch. A cardiac tamponade was successfully created. Injection of diluted contrast agent into the pericardial space via a catheter made it possible to clearly distinguish between the hemopericardium and the surrounding tissues on postmortem computed tomography. This procedure did not need an X-ray imaging system during catheterization. Our present model would help us examine the intrathoracic organs in the presence of acute cardiac tamponade.

Distribution of Severe Fever with Thrombocytopenia Syndrome Virus and Antiviral Antibodies in Wild and Domestic Animals in Oita Prefecture, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging life-threatening infectious disease caused by the tickborne SFTS virus (SFTSV), first identified in China in 2009 and then in Japan in 2013. Human SFTS cases were reported to be concentrated in western Japan, but the epidemiological conditions of SFTSV infection in a specific region are still obscure. We performed an epidemiological study of SFTSV in Oita Prefecture on the island of Kyushu, located in western Japan. For our research, we collected sera from wild and domestic animals (deer, wild boars, raccoons, cats, and dogs) and ticks from January 2010 to November 2020 in Oita. The anti-SFTSV antibody positivity rate of deer in 2014 was significantly higher than that in 2011 (65% versus 27%, P < 0.001). The anti-SFTSV antibody positivity rates of deer, wild boars, raccoons, wild dogs, domestic dogs, and domestic cats were 55%, 12%, 27%, 1.8%, 0.53%, and 1.4%, respectively. Moreover, RT-PCR could not detect SFTSV in any tick sample. Of the six areas of Oita Prefecture, only the Eastern area showed no incidence or possibility of SFTSV infection among wild and domestic animals, ticks, and human beings. Further investigation is required to assess whether local seroepidemiology in animals will help assess the risk of SFTSV infections in inhabitants.

Genetic population structure of invasive raccoons (Procyon lotor) in Hokkaido, Japan: Unique phenomenon caused by pet escape or abandonment.

Phylogeographic studies can resolve relationships between genetic population structure of organisms and geographical distributions. Raccoons have become feral in Japan, and in Hokkaido island, they have been rapidly increasing in number and spreading since the 1970s. We analyzed mitochondrial (mtDNA) and microsatellite DNA to understand the current phylogenetic distribution and invasive founder events. Overall, Hokkaido raccoons maintained high genetic diversity (i.e., the level of heterozygosity was comparable to the original habitat, North America). Based on mtDNA distribution and microsatellite diversity, Hokkaido raccoons were divided into six management units. However, mtDNA haplotype distributions and genetic structures based on microsatellites did not always correspond to each other (e.g., two geographically and genetically separated populations showed similar mtDNA distributions). In addition, a high degree of genetic admixture was observed in every unit, and the degree of genetic differentiation was low even between regions separated by long distances. Compared with other countries in Europe where genetic distribution of introduced raccoons is more clearly structured, the current results represent a unique and complex phenomenon of pet escape/abandonment in Hokkaido: i.e., genetically related colonies were introduced into multiple regions as founder events, resulting in the current state in which raccoons are not clearly genetically differentiated even 40 years after introduction.

Steroidogenic enzymes, their products and sex steroid receptors during testis development and spermatogenesis in the domestic cat (Felis catus).

In the present study we comprehensively characterize intratesticular sex steroid production, metabolism and receptors in the domestic cat to elucidate the role of testosterone, estradiol and progesterone in testis development, steroid synthesis and spermatogenesis. There is a great demand for new concepts of fertility control in domestic (feral) cats and wild felids. The acquired knowledge will help to understand the regulation of spermatogenesis in felids, and may reveal new target points for male contraception. Progesterone and androgens are produced throughout all stages of testicular development; their synthesizing enzymes are mainly expressed in Leydig cells, and to a much lesser extent also in tubular cells. Aromatase (CYP19A1), the estrogen synthesizing enzyme, is only present in the tubuli and is first detectable in spermatocytes and round spermatids at puberty. As shown by elevated expression of the enzymes steroid 5-α-reductase type 1 (SRD5A) and aldo-keto-reductase family 1 member C3 (AKR1C3), the capacity to metabolize particular steroids increases during testis development. Apparently, this refers to a decreasing intra-testicular testosterone concentration per mg tissue with increasing testis weight during postpuberty. The increasing potential of sulfation of E2 by estrogen sulfotransferase (SULT1E1) with ongoing development might be responsible for the low level of unconjugated intratesticular estradiol in all stages of development probably due to facilitated excretion of conjugated estrogens. For the first time, expression of the progesterone membrane receptor components 1 and 2 (PGRMC1, PGRMC2) was studied in mammalian testis tissue. Both of these and also the progesterone receptor (PGR) are expressed depending on the developmental stage and cell type, suggesting an important regulatory role of progesterone in the testis. Androgen receptor (AR) is present in almost all cell types except for some spermatogenic cells. The co-localization of aromatase with estrogen receptor alpha (ESR1) in spermatocytes and round spermatids of domestic cat testis indicates an auto-/paracrine function of estrogen in spermatogenesis. In summary, the testis of the domestic cat is an important source of sex steroids. All of them could act within the testis but additionally, at least androgens and estrogens are likely secreted by the testis, partly as conjugated steroids.

A transvaginal endoscopy-based technique for performing ovarian examinations in sows.

For examining pig ovaries, which have complex structures, laparoscopy is a useful technique, but requires general anesthesia; therefore, it cannot be performed repeatedly within a short period of time. We report a transvaginal endoscopy-based technique for conducting ovarian examinations without general anesthesia. Sows were sedated in pig stalls. Using a colonoscope, the vaginal wall was punctured with a trocar. To avoid the trocar being caught in the broad ligament of the uterus or the connective tissue around the vagina, the trocar was inserted close to the external uterine os and between the 2:00 and 3:00 or the 9:00 and 10:00 positions (in a clockwise direction). Then, a urethroscope was inserted into the abdomen, and an examination was carried out after the ovaries had been moved towards the urethroscope camera via rectal palpation. This less invasive procedure may allow repeated examinations and will increase our understanding of ovarian dynamics in pigs.

Comparative analysis of intraluteal steroidogenic enzymes emphasises the functionality of fresh and persistent corpora lutea during pro--and metoestrus in the lynx.

European lynx species demonstrate an atypical ovarian cycle compared to other felids. The physiological persistence of corpora lutea (CLs), reflected in constantly elevated progesterone (P4) concentrations in serum, is thought to ensure a seasonal monooestrus. Moreover, the coexistence of CLs from a recent ovulation (freshCLs) and persistent CLs from previous years (perCLs) on the same ovary has been proven. We assume that perCLs in lynxes occur due to fundamentally different mechanisms of luteal regression. Our study presents a detailed analysis of steroidogenic enzymes and steroids in fresh and perCLs obtained from Iberian lynxes during metoestrus, and in perCLs obtained from Eurasian lynxes during prooestrus. By quantitative PCR we measured relative mRNA amounts of steroidogenic acute regulatory protein (STAR), cytochrome P450 oxidases (CYPs), hydroxysteroid dehydrogenases (HSDs) and a steroid reductase (SRD). Protein expression in CLs was investigated for CYP11A1, CYP17A1, CYP19A1 and HSD3B. Additionally, the intraluteal and serum steroid content was determined. During metoestrus, mRNA amounts of STAR, CYP11A1, CYP19A1, HSD17B7 and SRD5A1 were significantly higher in perCLs compared to freshCLs. Protein of CYP11A1 was detected independently of the CL age in metoestrus, but expression was less evident in prooestrous perCLs. The protein signal of CYP17A1 was strong in freshCLs and perCLs of metoestrus, but weak at prooestrus. The presence of CYP19A1 protein was confirmed in each stage of the CL. These findings contribute to the hypothesis that CLs from previous years might support freshly developed CLs for pregnancy maintenance. However, initiation of ovulation might require a functional down-regulation of perCLs prior to breeding. It is noteworthy that the HSD3B1 mRNA amount was significantly elevated in fresh compared to perCLs (metoestrus). Accordingly, HSD3B protein was substantially present in freshCLs, whereas signals were literally absent in all perCLs. Elevated expression of HSD3B coincided with high intraluteal oestrogen concentrations in freshCLs; however, the enzyme pattern was less concordant with intraluteal P4 and androgen concentrations. Serum P4 concentrations of Iberian lynxes were constant between prooestrus and prolonged dioestrus. Moreover, constantly high serum oestrogen concentrations were measured during pro-, met- and prolonged dioestrus. The physiology of exceptionally high serum oestrogen concentrations outside the breeding season of lynxes merits further investigation. In conclusion our study supports the concept that the unique reproductive strategy of lynxes is directly linked to sustained intraluteal steroid biogenesis in persistent CLs.

Changes in the immunolocalization of steroidogenic enzymes and the androgen receptor in raccoon (Procyon lotor) testes in association with the seasons and spermatogenesis.

The raccoon is a seasonal breeder with a mating season in the winter. In a previous study, adult male raccoons exhibited active spermatogenesis with high plasma testosterone concentrations, in the winter mating season. Maintenance of spermatogenesis generally requires high testosterone, which is produced by steroidogenic enzymes. However, even in the summer non-mating season, some males produce spermatozoa actively despite low plasma testosterone concentrations. To identify the factors that regulate testosterone production and contribute to differences in spermatogenetic activity in the summer non-mating season, morphological, histological and endocrinological changes in the testes of wild male raccoons should be known. In this study, to assess changes in the biosynthesis, metabolism and reactivity of testosterone, the localization and immunohistochemical staining intensity of four steroidogenic enzymes (P450scc, P450c17, 3ßHSD, P450arom) and the androgen receptor (AR) were investigated using immunohistochemical methods. P450scc and P450c17 were detected in testicular tissue throughout the year. Seasonal changes in testosterone concentration were correlated with 3ßHSD expression, suggesting that 3ßHSD may be important in regulating the seasonality of testosterone production in raccoon testes. Immunostaining of P450arom and AR was detected in testicular tissues that exhibited active spermatogenesis in the summer, while staining was scarce in aspermatogenic testes. This suggests that spermatogenesis in the raccoon testis might be maintained by some mechanism that regulates P450arom expression in synthesizing estradiol and AR expression in controlling reactivity to testosterone.

A genetic method for sex identification of raccoons (Procyon lotor) with using the ZFX and ZFY genes.

A genetic method for sex determination in raccoons was developed based on nucleotide differences of the zinc finger protein genes ZFX and ZFY. Four novel internal primers specific for ZFX or ZFY were designed. PCR amplification using two primer sets followed by agarose gel electrophoresis enabled sex determination. 141-bp and 447-bp bands were in both sex, and 346-bp band was specific only in male with primer set I. 345-bp and 447-bp bands were in both sex, and 141-bp band was specific only in male with primer set II, which could distinguish raccoon's electrophoresis pattern from three native carnivores in Hokkaido. This method will be useful for conservation genetics studies or biological analyses of raccoons.

Timing of puberty and its relationship with body growth and season in male raccoons (Procyon lotor) in Hokkaido.

The raccoon (Procyon lotor), indigenous to North America, has naturalized in Japan as an invasive alien species, having been introduced into the country in the 1970s. In Hokkaido, the northernmost island of Japan, feral raccoons have been increasing in number and spreading throughout the island. The age at the onset of puberty for raccoons is important for estimating individual lifetime reproductive success and population growth. The present study investigated the timing of and potential factors affecting the onset of puberty in male raccoons in Hokkaido. External characteristics and histology of testes were studied in 151 male feral raccoons and in 1 captive juvenile. For the majority of feral yearling raccoons, prepubertal development began in May, and spermatozoa production began in October prior to their second mating season. However, some larger juveniles attained puberty during the juvenile period. The captive juvenile, which was fed throughout the winter, attained puberty only 11 months after birth. These results suggest that if male raccoons can achieve enough body growth before the first mating season, puberty can be attained early. In both juveniles and yearlings, spermatozoa production was only observed after autumn. This timing coincided with the recrudescence of seasonally active spermatogenesis in adult males. Therefore, attaining puberty in male raccoons appears to require both adequate body nutrient development and several environmental factors that control seasonal testicular changes.

Seasonal changes in spermatogenesis and peripheral testosterone concentration in raccoons (Procyon lotor) in Hokkaido.

Feral raccoons (Procyon lotor) have been increasing in number since 1979 and are currently subject to pest control in Hokkaido. One of the reasons for the increase in numbers is thought to be the high reproductive potential of raccoons, but little is known about their reproduction. The main aim of this study was to clarify seasonal changes in spermatogenesis and peripheral testosterone concentration of raccoons in Hokkaido. In the present study, external characteristics and histology of the testis and epididymis and the plasma testosterone concentration were investigated in 68 feral, male raccoons culled for pest control and once a month in one live, captive male. The feral males exhibited seasonal changes in spermatogenesis, showing active spermatogenesis in autumn, winter and spring (October-June) with noted spermatogenesis and inactive spermatogenesis in summer (July-September) with lower mean levels of spermatozoa in the cauda epididymis. Even in the inactive period, spermatozoa were observed in about half of the individuals (14/26); therefore, individuals producing spermatozoa existed every month throughout the year. Testosterone concentrations were significantly high in the winter mating season. In the captive male, the testosterone concentrations were low from June to August, and spermatozoa could not be observed from July to September. These results suggest that raccoons exhibit seasonality of reproduction, but the time and duration of spermatogenetic decline varies widely among individuals. This individual variation in the inactive period is a feature of male raccoon reproduction and is unique among seasonally breeding mammals.