Whole-genome Analyses Reveal Past Population Fluctuations and Low Genetic Diversities of the North Pacific Albatrosses.

Throughout the Plio-Pleistocene, climate change has impacted tropical marine ecosystems substantially, with even more severe impacts predicted in the Anthropocene. Although many studies have clarified demographic histories of seabirds in polar regions, the history of keystone seabirds of the tropics is unclear, despite the prominence of albatrosses (Diomedeidae, Procellariiformes) as the largest and most threatened group of oceanic seabirds. To understand the impact of climate change on tropical albatrosses, we investigated the evolutionary and demographic histories of all four North Pacific albatrosses and their prey using whole-genome analyses. We report a striking concordance in demographic histories among the four species, with a notable dip in effective population size at the beginning of the Pleistocene and a population expansion in the Last Glacial Period when sea levels were low, which resulted in increased potential coastal breeding sites. Abundance of the black-footed albatross dropped again during the Last Glacial Maximum, potentially linked to climate-driven loss of breeding sites and concordant genome-derived decreases in its major prey. We find very low genome-wide (π < 0.001) and adaptative genetic diversities across the albatrosses, with genes of the major histocompatibility complex close to monomorphic. We also identify recent selective sweeps at genes associated with hyperosmotic adaptation, longevity, and cognition and memory. Our study has shed light on the evolutionary and demographic histories of the largest tropical oceanic seabirds and provides evidence for their large population fluctuations and alarmingly low genetic diversities.

Non-Synonymous Substitutions in Cadherin 13, Solute Carrier Family 6 Member 4, and Monoamine Oxidase A Genes are Associated with Personality Traits in Thoroughbred Horses.

Retraining retired racehorses for various purposes can help correct behavioral issues. However, ensuring efficiency and preventing accidents present global challenges. Based on the hypothesis that a simple personality assessment could help address these challenges, the present study aimed to identify genetic markers associated with personality. Eight genes were selected from 18 personality-related candidate genes that are orthologs of human personality genes, and their association with personality was verified based on actual behavior. A total of 169 Thoroughbred horses were assessed for their tractability (questionnaire concerning tractability in 14 types of situations and 3 types of impressions) during the training process. Personality factors were extracted from the data using principal component analysis and analyzed for their association with single nucleotide variants as non-synonymous substitutions in the target genes. Three genes, CDH13, SLC6A4, and MAOA, demonstrated significant associations based on simple linear regression, marking the identification of these genes for the first time as contributors to temperament in Thoroughbred horses. All these genes, as well as the previously identified HTR1A, are involved in the serotonin neurotransmitter system, suggesting that the tractability of horses may be correlated with their social personality. Assessing the genotypes of these genes before retraining is expected to prevent problems in the development of a racehorse's second career and shorten the training period through individual customization of training methods, thereby improving racehorse welfare.

Rational determination of cefazolin dosage regimen in horses based on pharmacokinetics/pharmacodynamics principles and Monte Carlo simulations.

A pharmacokinetics/pharmacodynamics (PK/PD) approach was used to determine the best empirical dosage regimen of cefazolin (CEZ) after intramuscular (IM) administration of CEZ in horses. Seven horses received a single IM or intravenous (IV) administration of CEZ of 5 mg/kg bodyweight (BW) according to a crossover design. CEZ plasma concentrations were measured using LC-MS/MS. The plasma concentrations in these seven horses and those of six other horses obtained in a previous study with an IV CEZ dose of 10 mg/kg were modelled simultaneously using NonLinear Mixed-Effect modelling followed by Monte Carlo simulations to establish a rational dosage regimen. A 90% Probability of Target Attainment (PTA) for a PK/PD target of a free plasma concentration exceeding MIC90 (fT > MIC ) for 40% of the dosing interval was set for selecting an effective dosing regimen. The typical half-life of absorption and bioavailability after IM administration were 1.25 h and 96.8%, respectively. A CEZ dosage regimen of 5 mg/kg BW q12h IM administration achieved therapeutic concentrations to control both Streptococcus zooepidemicus and Staphylococcus aureus. For the same dose, the fT > MIC after IM administration was significantly longer than after IV administration, and the IM route should be favoured by clinicians for its efficiency and convenience.

Generic approach for the discovery of drug metabolites in horses based on data-dependent acquisition by liquid chromatography high-resolution mass spectrometry and its applications to pharmacokinetic study of daprodustat.

In drug metabolism studies in horses, non-targeted analysis by means of liquid chromatography coupled with high-resolution mass spectrometry with data-dependent acquisition (DDA) has recently become increasingly popular for rapid identification of potential biomarkers in post-administration biological samples. However, the most commonly encountered problem is the presence of highly abundant interfering components that co-elute with the target substances, especially if the concentrations of these substances are relatively low. In this study, we evaluated the possibility of expanding DDA coverage for the identification of drug metabolites by applying intelligently generated exclusion lists (ELs) consisting of a set of chemical backgrounds and endogenous substances. Daprodustat was used as a model compound because of its relatively lower administration dose (100 mg) compared to other hypoxia-inducible factor stabilizers and the high demand in the detection sensitivity of its metabolites at the anticipated lower concentrations. It was found that the entire DDA process could efficiently identify both major and minor metabolites (flagged beyond the pre-set DDA threshold) in a single run after applying the ELs to exclude 67.7-99.0% of the interfering peaks, resulting in a much higher chance of triggering DDA to cover the analytes of interest. This approach successfully identified 21 metabolites of daprodustat and then established the metabolic pathway. It was concluded that the use of this generic intelligent "DDA + EL" approach for non-targeted analysis is a powerful tool for the discovery of unknown metabolites, even in complex plasma and urine matrices in the context of doping control.

Concentration of cephalothin in body fluids and tissue samples of Thoroughbred horses.

Cephalothin (CET) concentrations in body fluids (plasma, synovial fluid, pleural fluid, peritoneal fluid, and aqueous humor) and tissue samples (bone, lung, jejunum, hoof, and subcutaneous tissue) were investigated to consider the treatment of infectious diseases in horses. CET 22 mg/kg body weight was intravenously administered to 12 horses. Samples were collected from four different horses at 1, 3, and 5 hr after administration. The CET concentration in body fluids other than aqueous humor was maintained above the MIC90 values of Streptococcus zooepidemicus and Staphylococcus aureus until 5 hr, but it was not maintained above that of S. aureus in bone. CET (22 mg/kg twice a day) is effective for septic arthritis, pleuritis, and peritonitis caused by gram-positive bacteria but ineffective for osteomyelitis.

Effect of advancing age on the reproductive performance of Japanese Thoroughbred broodmares.

Many studies have reported that advancing age in broodmares has a negative impact on the reproductive performance of horses. However, although the ages at first and last mating vary among broodmares, it is unknown how this variation affects the correlation between age and reproductive performance in mares. Here, in order to examine the effects of the ages at first and last mating, we analyzed all recorded mating events for Thoroughbreds in Japan from 1997 to 2017. We found that the live foal birth rate of mares with a younger age at first mating indeed declined at an earlier age than those with an older age at first mating and that the number of years since the first mating also contributes to the decline in the birth rate. We also found that the live foal birth rate and mean earnings of the produced foals are much higher for mares with an older age at last mating compared with mares with a younger age at last mating. Our results should aid breeders in assessing the value of broodmares and designing breeding strategies.

Influence of exercise and emotional stresses on secretion of prolactin and growth hormone in Thoroughbred horses.

The secretion of prolactin and growth hormone in response to exercise and emotional stresses was investigated in Thoroughbreds. Two experiments were performed: one with loading of only exercise stress and one with simultaneous loading of exercise and emotional stresses. Exercise stress was loaded in 4 steps using a treadmill for horses: pre-exercise period (5 min), walking period (6.5 min), galloping period (3 min), and cooling down period (10 min). Emotional stress was loaded by showing a loud video of an audience at a racetrack during the walking period. The results clearly demonstrated that exercise stress rapidly increased the secretion of prolactin and growth hormone and that secretion of them persisted for a specific period after the exercise. In addition, emotional stress promoted prolactin secretion.

Equine nonneoplastic abnormal ovary in a draft mare with high serum anti-Müllerian hormone: a case study.

We performed a standing hand-assisted laparoscopic ovariectomy in a draft mare that presented with high serum anti-Müllerian hormone (AMH) level and had an enlarged single cystic ovary. Histopathological examination revealed no tumor cell proliferation in the ovary, but the presence of a large ovarian cyst was confirmed. In the diagnosis of abnormal ovaries in mares, a comprehensive assessment should be performed, including the monitoring of ovarian morphology and biomarkers over time, to determine the disease prognosis and treatment plan. The case of this mare with a nonneoplastic abnormal ovary and increased serum AMH level was rare. We suggest that standing hand-assisted laparoscopic ovariectomy is useful for the removal of large ovaries in draft mares.

Changes of lying behavior in Thoroughbred foals influenced by age, pasturing time, and weather conditions.

The lying behavior of Thoroughbred foals on breeding farms was continuously measured using triaxial accelerometers. Accelerometers were attached on the side of the hind limb cannon and under the halter of six foals to record triaxial angle data every 10 sec for a period of 24 hr. Lying behavior was divided into sternal lying and lateral lying based on head angle. Sampling was performed for two consecutive days each week until weaning. Sampling periods were divided into two periods on the basis of pasturing time: daytime pasturing (period A: 7-hr pasturing period, 2-60 days of age) and overnight pasturing (period B: 19-hr pasturing period, 32-152 days of age). Lying duration and frequency were longer and higher, respectively, in period A (44.6% of the time and 26.8 ± 7.4 times per day) than those in period B (27.7% of the time and 15.3 ± 4.2 times per day). In addition, foals spent more time laterally in period A (48.1% of total lying time) than in period B (38.9% of total lying time). Foals lie down longer in stalls than in pastures (Period A, 56.3 vs. 16.0%; Period B, 52.5 vs. 21.1%). Lateral lying was also longer in the stall than in the pasture. Lying duration and frequency changed with age in period A. Wet weather prevented lying behavior in the pasture. These results suggested that age, weather, and pasturing time affected the lying behavior of Thoroughbred foals under management at a breeding farm. Triaxial accelerometers may be useful for monitoring equine lying behavior.

Expression of activin receptors in the equine uteroplacental tissue: an immunohistochemical analysis.

Activin is secreted from equine uterine glands and plays important roles in establishment and maintenance of pregnancy in mares. This study aimed to localize activin receptors (ActRs) IA/B and IIA/B using immunohistochemistry in the uteroplacental tissues of seven pregnant Thoroughbred mares. At the time of tissue collection, the mares were at the following days of pregnancy: 88, 120, 161, 269, 290, 313, and 335 days. We fixed the uteroplacental tissues in 4% paraformaldehyde and obtained serial sections that were subsequently stained for analysis. All four isoforms of ActR were expressed in the uteroplacental tissues, including the endometrial epithelium, uterine glands, trophoblasts, and myometrium, throughout pregnancy. Our results suggested the potential role of activin in the uteroplacental tissues.

Application of a wearable GPS unit for examining interindividual distances in a herd of Thoroughbred dams and their foals.

Global positioning system (GPS) units are now lightweight and compact. They have proven useful for analyzing the behavioral characteristics of horses in pastures. Because the GPS records data in latitude and longitude, it may be feasible to calculate the distance between GPS units. The present study aimed to confirm the applicability of GPS units in behavioral studies on horses. For this, we analyzed the accuracy of the distances calculated from GPS units using Hubeny's distance formula and of the monthly changes in interindividual distances obtained from GPS units worn by Thoroughbred dams and their foals in a pasture until weaning. The calculated inter-GPS distances were highly accurate. The regression line was linear, and the squared correlation coefficient (r2) was 0.9998. During the first month of age, the interindividual dam-dam and foal-foal distances were significantly greater than the dam-foal distance. During the second month of age, the dam-foal distance increased once and gradually decreased up to the fifth month of age. During the sixth month of age, the dam-foal distance was significantly greater than the foal-foal distance. The GPS distances calculated using Hubeny's distance formula were useful for analyzing changes in interindividual distances in a herd of Thoroughbred dams and their foals. Most likely, calculation of the distance between GPS units worn on equine head collars is likely to become a very useful tool as an objective index for quantifying equine behavioral observations.

Histopathological findings of apical fracture of the proximal sesamoid bones in young Thoroughbred foals.

Although radiographic findings at the apical portion of the proximal sesamoid bone (PSB) are often observed in young Thoroughbred foals, conflicting findings, either fractures or apparent secondary ossifications centers, have been reported. Three cases (aged 2, 5, and 7 weeks) were identified in 30 necropsied foals (0-31 weeks old). Histopathologically, the subchondral trabecular woven bone was fractured and exhibited focal necrosis of woven bone, fibrin exudate, and/or fibrosis within the foci. In the 7-week-old case, proliferations of chondrocytes were also observed. These findings suggest that the radiographic findings represented a healing process of the apical PSB fractures associated with the mechanically damaged subchondral trabeculae. Developmental PSB injuries should be taken into consideration during the management of young Thoroughbred foals.

Aborted fetal sizes of Thoroughbred horses in Hidaka, Japan, between 2005 and 2015.

The degree of fetal growth restriction has been unclear in equine reproduction. In this study, 2,195 fetuses from 2,137 abortions during 11 seasons were examined to determine the causes of abortion, and fetal size dimensions (crown rump length and body weight) were measured. In total, 900 cases (42.1%) of abortion were identified as caused by viral infection (215, 10.1%), bacterial infection (156, 7.3%), fungal infection (25, 1.2%), circulation failure (406, 19.0%), multiple causes (66, 3.1%), deformity (13, 0.6%), placental abnormality (12, 0.6%), and other causes (7, 0.3%). All viral infections originated from equine herpes virus. Of all abortions, 94.3% occurred between 181-360 days of pregnancy, and the gestational ages at abortion were different based on the causes. Fetal sizes in viral abortions were considerably larger than those due to other reasons. Compared with viral infection, the crown rump length size dimension of fetuses aborted from multiple and fungal infection was affected. In addition, bacterial infection, circulation failure, and unknown causes of abortions also contributed to growth restriction in terms of body weight. In conclusion, the present study showed details of equine abortion and the relationships between causes of abortion and fetal size. Most of the aborted fetuses showed restrictions in their growth. The manifestations of growth restriction were more related to weight than skeletal length.

Comparison of physical body growth and metabolic and reproductive endocrine functions between north and south climates of Japan in trained Thoroughbred yearling horses.

This study aimed to compare body growth, metabolic, and reproductive hormonal changes in trained Thoroughbred yearling horses under different climate conditions with and without light supplementation (LS). Thoroughbred yearlings raised at research centers of the Japan Racing Association in Hokkaido (north) or Miyazaki (south) were divided into control and LS groups. In the LS groups, 44 colts and 47 fillies from Hokkaido and 11 colts and 11 fillies from Miyazaki were exposed to LS with an extended photoperiod of 14.5 hr of daylight and 9.5 hr of darkness. One week before and once a month after LS, circulating total thyroxine (T4), insulin-like growth factor-I (IGF-1), prolactin (PRL), cortisol, and progesterone (P4) concentrations were measured by radioimmunoassay and fluoroimmunoassay, respectively. Growth parameters, including body weight, height, girth, and cannon bone circumferences, were measured monthly. Hair coat (HC) condition was scored. Under natural conditions, the T4 concentrations of Hokkaido yearlings tended to be higher, whereas the IGF-1 (colt) and PRL levels were significantly lower than those of yearlings in Miyazaki. Growth parameters and HC scores were lower in Hokkaido yearlings. With LS, the PRL and P4 concentrations in Hokkaido and Miyazaki were higher, and the first ovarian activity tended to be earlier than in the controls. Only LS Hokkaido yearlings showed significantly higher HC scores than the controls. Comparing the different climates among the LS yearlings, the levels of PRL and P4 and the HC scores in Hokkaido yearlings increased and reached levels similar to those in Miyazaki yearlings. The body weight and girth increment percentages of Hokkaido yearlings in January dramatically decreased and then eventually increased to levels similar to those of Miyazaki yearlings. This suggested that yearlings in naturally colder Hokkaido exhibit higher basal metabolism to maintain homeostasis. However, providing LS may help to improve growth and early development of reproductive function in Hokkaido yearlings to levels equal to those of Miyazaki horses.

Characteristic findings of magnetic resonance imaging (MRI) and computed tomography (CT) for severe chronic laminitis in a Thoroughbred horse.

A Thoroughbred horse with severe chronic laminitis of both forelimbs was evaluated on the same day with magnetic resonance imaging (MRI) and computed tomography (CT). Both MRI and CT revealed loss of the dorsal aspect of the cortical bone of the 3rd phalanx and sclerosis. CT reflected the status of the horny layer and bone of the affected feet, while MRI depicted inflammation of the laminar corium, together with tendon edema. On the 3-dimensional CT venogram, vessels were visualized in both the right and left forelimbs, although there was a difference in the vasculature of the coronary plexus and circumflex vessels between the right and left forelimbs. A combination of both MRI and CT provides detailed information regarding pathological conditions.

Foaling rate of mares that were rebred after pregnancy loss in Hidaka, Japan.

The purpose of this study was to evaluate foaling rates of mares that were rebred after the pregnancy loss in same reproductive season and to examine factors influencing them in Hidaka, Japan. The study included 82 Thoroughbred mares that had experienced pregnancy loss. The foaling rate of the mares that were rebred after pregnancy loss was 57.3%. The foaling rate decreased as the period until detection of pregnancy loss increased. Aging and lower body condition score of mares decreased the foaling rate.

Sequence variants of BIEC2-808543 near LCORL are associated with body composition in Thoroughbreds under training.

Ligand-dependent nuclear receptor compressor-like (LCORL) encodes a transcription factor, and its polymorphisms are associated with measures of skeletal frame size and adult height in several species. Recently, the single nucleotide polymorphism (SNP) BIEC2-808543 located upstream of LCORL was identified as a genetic diagnostic marker associated with withers height in Thoroughbreds. In this study, 322 Thoroughbreds-in-training were genotyped for BIEC2-808543 to evaluate the association between genotype and body composition traits, including body weight, withers height, the ratio of body weight to withers height, chest circumference, and cannon circumference. Of these, withers height and cannon circumference were significantly associated with LCORL genotypes throughout almost the entire training period in males and females. Animals with a C/T genotype had higher withers height (maximum differences of 1.8 cm and 2.1 cm in males and females, respectively) and cannon circumstance (maximum differences of 0.65 cm and 0.48 cm in males and females, respectively) compared with animals with a T/T genotype. These results suggested that the regulation of LCORL expression influences the skeletal frame size in Thoroughbreds and thus, indirectly affects the body weight. Although LCORL and BIEC2-808543 would be useful for selective breeding in Thoroughbreds, the production of genetically modified animals and gene doping based on genetic information should be prohibited in order to maintain racing integrity.

Autopsy imaging for cardiac tamponade in a Thoroughbred foal.

Autopsy imaging (Ai), postmortem imaging before necropsy, is used in human forensic medicine. Ai was performed using computed tomography (CT) for a 1-month-old Thoroughbred foal cadaver found in a pasture. CT revealed pericardial effusion, collapse of the aorta, bleeding in the lung lobe, gas in the ventricles and liver parenchyma, and distension of the digestive tract. Rupture in the left auricle was confirmed by necropsy; however, it was not depicted on CT. Therefore, Ai and conventional necropsy are considered to complement each other. The cause of death was determined to be traumatic cardiac tamponade. In conclusion, Ai is an additional option for determining cause of death.

Quantitative evaluation of cervical cord compression by computed tomographic myelography in Thoroughbred foals.

Five Thoroughbred foals (age, 8-33 weeks; median age, 31 weeks; weight, 122-270 kg; median weight, 249 kg) exhibiting ataxia with suspected cervical myelopathy (n=4) and limb malformation (n=1) were subjected to computed tomographic (CT) myelography. The areas of the subarachnoid space and cervical cord were measured on transverse CT images. The area of the cervical cord was divided by the area of subarachnoid space, and stenosis ratios were quantitatively evaluated and compared on the basis of histopathological examination. The sites with a ratio above 52.8% could have been primary lesion sites in the histopathological examination, although one site with a ratio of 54.1% was not a primary lesion site. Therefore, in this study, a ratio between 52.8-54.1% was suggested to be borderline for physical compression that damages the cervical cord. All the cervical vertebrae could not be scanned in three of the five cases. Therefore, CT myelography is not a suitable method for locating the site of compression, but it should be used for quantitative evaluation of cervical stenosis diagnosed by conventional myelography. In conclusion, the stenosis ratios determined using CT myelography could be applicable for detecting primary lesion sites in the cervical cord.

A clinical case of equine fungal placentitis with reference to hormone profiles and ultrasonography.

Fungal placentitis is an infectious disease inducing abortion in pregnant mares. In the present report, we describe a field case of abortion caused by fungal placentitis with consecutive examinations. The progesterone level and combined thickness of the uterus and placenta (CTUP) were abnormal before the onset of clinical signs. Additionally, the estradiol level started to change before the appearance of clinical signs. Abnormal serum amyloid A values and an abnormal fetal heart rate were observed after the onset of clinical signs. The present report demonstrates that the progesterone level and CTUP may be adequate as early diagnostic markers of fungal placentitis and bacterial infection. Endocrinological evaluation based on cutoff values or serial measurements were also useful for early diagnosis.