Genome-wide search reveals the uniqueness of DNA regions associated with coat color and innate immunity in Hokkaido Native Horse.

Hokkaido Native Horse (HKD) is a horse breed native to Hokkaido in Japan known for the traits such as coat color with no white spots and adaptability to the local cold climate. To examine whether those traits of HKD are conferred at the DNA level, we attempted to identify fixed DNA regions in HKD individuals, that is, the selection signatures of HKD. A comparison of genome-wide single nucleotide polymorphism genotypes in 58 HKD individuals by principal component analysis, and cluster analysis between breeds, including HKD, and within the HKD individuals indicated the genetic independence of HKD as a breed. Tajima's D analysis and runs of homozygosity analysis identified 23 selection signatures unique to HKD (P < 0.05), and following database search found 20 traits that were associated with those selection signatures; among these traits, coat color traits, face and body markings, showed the highest important value (0.50 and 0.46). Enrichment analysis of genes in the selection signatures identified six gene ontology terms (P < 0.05), and a term related to innate immunity (regulation of defense response; GO:0031347) showed the highest positive fold enrichment value (7.13). These results provide the first scientific evidence of a genetic basis for the traits of HKD.

Maternal effect on first-year growth of Hokkaido native foals kept outdoors all year round.

In this study, we investigated maternal effects on the growth of Hokkaido native horses kept outdoors throughout the year. We used the data on body weight (BW), height at withers (HW), heart girth (HG), and cannon circumference (CC) of 517 foals during the first year of life to (1) examine the growth patterns by comparing six linear mixed models and (2) analyze the maternal effect on BW, HW, HG, and CC for each month by estimating variance components. The segmented polynomial third-order regression equation was selected as the best model for all the traits. The estimated proportion of variance components for the effects of the dam were 0.25-0.38 for BW, 0.19-0.28 for HW, 0.19-0.34 for HG, and 0.10-0.21 for CC. A higher effect of the dam compared with that of the sire was observed on BW and HG at all months of age and HW at 0-2 months of age. Therefore, the dam contributed to foal growth not only through half of its heredity but also through the maternal effect derived mainly from its milk. These results provide valuable information for the breeding of Hokkaido native foals.

Relationship between foal daily gain, suckling behavior, and the distance between foals and their mares in the first 40 days of life of Hokkaido native horses kept outdoors year-round.

In this study, we investigated suckling behavior and the distance between a foal and its mare as indicators of foal growth in Hokkaido native horses (HKD) during the first 40 days of life. Direct observations were made of 22 mare and foal pairs from two consecutive days 1 and 2, 5 and 6, 10 and 11, 20 and 21, 30 and 31, and 40 and 41 after birth by focal animal sampling for 3 h per day. Suckling behavior was continuously recorded, along with the terminator. The distance between the foal and its mare was determined using the instantaneous sampling method (1 min), and the percentage was calculated within 4 m while the foal was recumbent (DPW4FR). In the first week after birth, the duration and frequency of suckling terminated by the foal were negatively correlated with foal daily gains (duration, -0.49, p = 0.028; frequency, -0.53, p = 0.017). The DPW4FR and daily gain during 10-40 days after birth showed significant negative correlation (-0.47, p = 0.031). It is suggested that in HKD reproduction systems, suckling behavior termination by the foal and the distance between the foal and mare may be indicators of early foal growth.

Effects of grazing adaptation on herbage intake, milk production, and body weight change in lactating dairy cows after turning out to pasture in early spring.

The present study investigated the effects of adaptation to grazing in early spring on the herbage intake, milk production, and body weight changes in lactating dairy cows. The experiment included 12 Holstein lactating cows during early spring. Six cows were allowed to gradually adapt to grazing (ADP) over 10 days. They were allowed to graze on a pasture for 4 h during the first week and for 19 h during the remaining 3 days (adaptation period). During the 10-day period, the remaining six cows were housed in a barn (CON). Cows in both groups received adequate silage, hay, and concentrate during the adaptation period. Subsequently, cows in both groups were allowed to graze together for 19 h for 4 weeks (experimental period). No cow received supplements during the experimental period. In the first week of the experimental period, the ADP cows showed a higher herbage intake than the CON cows. During the subsequent weeks, there were no differences in herbage intake between the two groups. At the start of the experimental period, milk production and body weight of the ADP cows were higher than those of the CON cows, and this difference lasted until the end of the experiment.

Changes of sires in a breeding farm enables maintenance of DNA-level genetic variation in a produced herd of Hokkaido Native Horses.

We investigated whether regular changes of the sire in a breeding farm of Hokkaido Native Horses (HKDs) enables the DNA-level genetic variation of the produced animals to be maintained. The genotypes of 31 microsatellite markers were identified and analyzed in 207 animals produced in a breeding farm in which the sire was replaced every 3 to 5 years. The mean allele number indicating the degree of genetic variation was 5.97 and was similar to those reported previously. The mean observed heterozygosity was 0.74 and was higher than the expected heterozygosity, 0.69; FIS was -0.07, indicating that the analyzed animals reflected frequent outbreeding and had maintained genetic variation. Based on genetic structural analysis, the number of genetic subpopulations of the animals was estimated to be as 6, and the majority (more than 50%) of each subpopulation corresponded to the progeny of one of the sires used in the breeding farm; these observations suggested that genetic variation in the analyzed animals reflected the genetic differences among sires. Pedigree records indicated that the average co-ancestry coefficient between sires used in the breeding farm was 0.015 corresponding to second cousin. This level of kinship among sires is acceptable for producing HKDs that maintain genetic variation.

Trophectoderm regeneration to support full-term development in the inner cell mass isolated from bovine blastocyst.

Which comes first: tissue structure or cell differentiation? Although different cell types establish distinct structures delineating the inside and outside of an embryo, they progressively become specified by the blastocyst stage, when two types of cell lineages are formed: the inner cell mass (ICM) and the trophectoderm (TE). This inside-outside aspect can be experimentally converted by the isolation of the ICM from a blastocyst, leading to a posteriori externalization of the blastomeres composing the outermost layer of the ICM. Here, we investigated the totipotency of isolated mouse and bovine ICMs to determine whether they are competent for TE regeneration. Surprisingly, a calf was generated from the bovine isolated ICM with re-formed blastocoel (re-iICM), but no mouse re-iICMs developed to term. To further explore the cause of difference in developmental competency between the mouse and bovine re-iICMs, we investigated the SOX17 protein expression that is a representative molecular marker of primitive endoderm. The localization pattern of SOX17 was totally different between mouse and bovine embryos. Particularly, the ectopic SOX17 localization in the TE might be associated with lethality of mouse re-iICMs. Meanwhile, transcriptome sequencing revealed that some of the bovine re-iICMs showed transcriptional patterns of TE-specific genes similar to those of whole blastocysts. Our findings suggest that TE regeneration competency is maintained longer in bovine ICMs than in mouse ICMs and provide evidence that the ICM/TE cell fate decision is influenced by structural determinants, including positional information of each blastomere in mammalian embryos.

Are Horses (Equus caballus) Sensitive to Human Emotional Cues?

Emotions are important for social animals because animals' emotions function as beneficial cues to identify valuable resources such as food or to avoid danger by providing environmental information. Emotions also enable animals to predict individuals' behavior and determine how to behave in a specific context. Recently, several studies have reported that dogs are highly sensitive to not only conspecific but also human emotional cues. These studies suggest that domestication may have affected such sensitivity. However, there are still few studies that examine whether other domesticated animals, in addition to dogs, exhibit sensitivity to human emotional cues. In this study, we used a gaze-following task to investigate whether horses (Equus caballus) are sensitive to human emotional cues (happy, neutral, disgust) and if they adjust their behavior accordingly. In the study, the experimenter suddenly turned her head to either right or left and showed emotional cues. The results revealed that horses significantly decreased the frequency with which they followed the experimenter's gaze and the total looking time during the gaze-emotional cue presentation in the Disgust condition compared to the Neutral condition. These results suggest the possibility that horses are sensitive to human emotional cues and behave on the basis of the meaning implied by negative human emotional cues.

Occurrence and seasonal variation of equine estrogens, equilin and equilenin, in the river water of Japan: Implication with endocrine-disrupting potentials to Japanese medaka (Oryzias latipes).

In this study, we determined the concentration of equine estrogens, such as equilin (Eq) and equilenin (Eqn), in the river water collected from nine research stations in Hokkaido, Japan. The LC-MS/MS analysis revealed that Eq concentrations were 2.7 ±â€¯6.7, 0.22 ±â€¯0.12, and 1.2 ±â€¯0.64 ng/L in Sep 2015, Feb 2016, and Jul 2016, respectively. Eqn had concentration levels similar to those of Eq. Comparison of the concentrations at nine research stations showed that seasonal variation was observed in the detected Eq and Eqn concentration levels. This study was the first to show the occurrences and seasonal variation of Eq and Eqn in the river water of Japan. We further investigated the reproductive and transgenerational effects of Eq in Japanese medaka (Oryzias latipes) exposed to 10, 100, and 1000 ng/L for 21 days and assessed the transcriptional profiles of the estrogen-responsive genes in the livers of both sexes. The reproduction assay demonstrated that 1000 ng/L of Eq adversely affected the reproduction (i.e. fecundity) in the F0 generation and that the hatching of F1 generation fertilized eggs was reduced in the 100 and 1000 ng/L treatment groups. Our qRT-PCR assay revealed that the mRNA expression levels of hepatic vitellogenin 1 and 2, choriogenin L and H, and estrogen receptor α were significantly up-regulated in males exposed to 100 and/or 1000 ng/L of Eq. In contrast, the transcriptional levels of several genes, such as pregnane X receptor and cytochrome P450 3A, were down-regulated in the livers of males after the 21-d exposure. These results suggest that Eq has endocrine-disrupting potential such as reproductive and transgenerational effects by the modulation of hepatic estrogen-responsive genes expression on medaka.

Accelerated invagination of vacuoles as a stress response in chronically heat-stressed yeasts.

When exposed to sublethal high temperatures, budding yeast cells can survive for a period of time; however, a sufficient amount of ubiquitin is necessary for this survival. To understand the nature of the stress, we examined the morphological changes in yeast cells, focusing on the vacuoles. Changes in vacuolar morphology were notable, and ruffled vacuolar membranes, accelerated invaginations of vacuolar membranes, and vesicle-like formations were observed. These changes occurred in the absence of Atg1, Atg9 or Ivy1 but appeared to require endosomal sorting proteins, such as Vps23, Vps24 or Pep12. Furthermore, the serial sections of the vacuoles analysed using an electron microscopic analysis revealed that spherical invaginated structures were linked together in a vacuole. Because degradation of cell surface proteins is induced from heat stress, fusion of endosomal and vacuolar membranes might occur frequently in heat-stressed cells, and yeast cells might be able to cope with a rapid increase in vacuolar surface area by such invaginations.

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.