Ovarian dynamics in progesterone tablet-induced superovulation in goats assessed by magnetic resonance imaging.

Controlled internal drug-releasing (CIDR) devices are commonly used for superovulation in goats. However, such devices are unavailable in some countries, including Japan. In this technical note, we aimed to explore the efficacy of an alternative superovulation protocol using progesterone tablets in goats. We employed intravaginal progesterone tablets (LUTINAS® Vaginal Tablet 100 mg) following a standard superovulation protocol. Additionally, we assessed the ovarian dynamics using 3T-magnetic resonance imaging (MRI) 1 day preceding the progesterone treatment (Day "-1") and 3 days before the end of treatment (Days 11-13). The ovarian monitoring was successfully performed in the short tau inversion recovery T2-weighted images of MRI, and ovulation was confirmed by the disappearance of follicles on Day 13 post-administration of the tablets. Immediately after ovulation, oviduct flushing yielded a substantial number of oocytes (13.5 ± 1.8 oocytes per animal). These findings provide evidence that the administration of progesterone tablets can serve as a viable alternative for inducing. Additionally, our findings suggest that 3T-MRI is a promising alternative to conventional ultrasonography for monitoring ovarian dynamics following superovulation in experimental goats.

Changes in population structure and genetic diversity of Misaki horses between 2015 and 2020.

For the preservation of Misaki horses, changes in the population structure and genetic diversity of the horses for 5 years were analyzed using population and genotype data from 2015-2020. The microsatellite genotyping was performed, and the average number of alleles (Na), expected heterozygosity (He), and observed value (Ho) were calculated. Moreover, the average generation length (GL) was estimated from the population management record. Then, no significant differences in Na, He, and Ho were found between 2015 and 2020, suggesting their genetic diversity had been maintained for 5 years. Moreover, the average GL was estimated as 4.6 years. Compared to other native horses, a short average GL suggesting a rapid generation renewing is a characteristic of the Misaki population.

Genetic characterization of Japanese native horse breeds by genotyping variants that are associated with phenotypic traits.

Concerns have been raised about the loss of genetic diversity in Japanese native horses because of their declining populations. In this study, we investigated the genetic variation of four genes, myostatin (MSTN), ligand-dependent nuclear receptor corepressor like (LCORL), doublesex and mab-3 related transcription factor 3 (DMRT3), and 5-hydroxytryptamine receptor 1A (HTR1A), which are associated with horse phenotypic traits, in six Japanese horse breeds (Hokkaido, Kiso, Noma, Misaki, Tokara, and Yonaguni). MSTN, LCORL, DMRT3, and HTR1A showed polymorphisms in the Kiso; Hokkaido and Noma; Hokkaido; and Kiso, Tokara, and Yonaguni breeds, respectively. The Misaki did not show polymorphisms in any of the genes. This study may serve as a basis for developing future breeding strategies focusing on traits in Japanese native horses.

Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control.

The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations in this study. Four models of gene editing (three homoALT with 1-bp insertions, one REF/ALT with 77-bp deletion) were constructed by editing the myostatin gene in horse fibroblasts using CRISPR/Cas9. The edited cells and 101 samples from thoroughbred horses were screened using the developed test, which was capable of identifying the three homoALT cells containing 1-bp insertions. Furthermore, 147 SNVs were investigated for their utility in confirming biological parentage. Of these, 120 SNVs were amenable to consistent and accurate genotyping. Surrogate (nonbiological) dams were excluded by 9.8 SNVs on average, indicating that the 120 SNV could be used to detect foals that have been produced by somatic cloning or embryo transfer, two practices that are prohibited in thoroughbred racing and breeding. These results indicate that gene-editing tests that include variant calling and SNV genotyping are useful to identify genetically modified racehorses.

Sequence determination of phosphorothioated oligonucleotides using MALDI-TOF mass spectrometry for controlling gene doping in equestrian sports.

In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model of therapeutic oligonucleotides, 22 bp-long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short-length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 104 (standard deviation: 4.34 × 103 ) was detected from 500 pmol PSO in 1 ml horse plasma using the linear negative mode of MALDI-TOF MS. In addition, a 17 bp sequence was determined using in-source decay (ISD) mode, indicating that 500 pmol of a PSO in 1 ml plasma is the detection limit for sequencing. Using the determined sequences (17 bp), a targeted gene for PSO was singly identified on the horse reference genome, EquCab2.0, via a GGGenome search. These procedures can be potentially used to identify therapeutic oligonucleotides, whose nucleotides are unknown, for gene doping control.

Physiological variation in Japanese macaques (Macaca fuscata) housed in different outdoor cages evaluated using the metabolic profile test.

Captive primates require environmental enrichment to minimize physical and mental stress. However, only a few objective evaluations have been performed to assess environment-induced physiological variations in these animals. In this study, we evaluated the usage of the metabolic profile test (MPT) to assess the influences of the housing environment on the physiology of Japanese macaques. Five male macaques were housed in an old type of cage (old cage group), in which the macaques were exposed to wind (except for a shelter box), and four males were in a new -type of cage (new cage group), which had a sub-room with a waterer that was surrounded by insulating panels. Blood samples were collected bimonthly for a year from the two groups to determine the complete blood count and blood biochemistry. The increase in the body weight of the macaques in both groups was suppressed during the cold season. Furthermore, this suppression was more pronounced in the old cage group, suggesting that the energy expenditure was higher in the old cage group than in the new cage group. Moreover, the red blood cell count and hematocrit values were higher during the cold season in both cages than during the warm season, suggesting that macaques were dehydrated during the cold season. Dehydration tendency was more pronounced in the macaques from the old cage group than in those from the new cage group, suggesting that their water intake decreased during the cold season. Our results suggest that the MPT can be used to evaluate environment-induced physiological variations in Japanese macaques.

Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control.

Gene doping has raised concerns in human and equestrian sports and the horseracing industry. There are two possible types of gene doping in the sports and racing industry: (1) administration of a gene-doping substance to postnatal animals and (2) generation of genetically engineered animals by modifying eggs. In this study, we aimed to identify genetically engineered animals by whole-genome resequencing (WGR) for gene-doping control. Transgenic cell lines, in which the erythropoietin gene (EPO) cDNA form was inserted into the genome of horse fibroblasts, were constructed as a model of genetically modified horse. Genome-wide screening of non-targeted transgenes was performed to find structural variation using DELLY based on split-read and paired-end algorithms and Control-FREEC based on read-depth algorithm. We detected the EPO transgene as an intron deletion in the WGR data by the split-read algorithm of DELLY. In addition, single-nucleotide polymorphisms and insertions/deletions artificially introduced in the EPO transgene were identified by WGR. Therefore, genome-wide screening using WGR can contribute to gene-doping control even if the targets are unknown. This is the first study to detect transgenes as intron deletions for gene-doping detection.

Decreased genetic diversity in Kiso horses revealed through annual microsatellite genotyping.

The Kiso horse is native to Japan and is on the verge of extinction. Here, we used microsatellites to characterize changes in their genetic diversity over time. We divided a population of Kiso horses that genotyped during 2007-2017 into three groups based on birth year: Group 1, 1980-1998 (70 horses); Group 2, 1999-2007 (61 horses); and Group 3, 2008-2017 (42 horses). We genotyped 31 microsatellites to calculate average number of alleles, observed heterozygosity, and expected heterozygosity. All indicators decreased across age groups. The results indicate that Kiso horses have been experiencing a drop in genetic diversity, and the population is expected to experience further decline unless appropriate measures are implemented.

Genetic characteristics of feral Misaki horses based on polymorphisms of microsatellites and mitochondrial DNA.

The Misaki horse is a Japanese native horse, known as the "feral horse of Cape Toi". In this study, we acquired the genetic information to establish their studbook, and analyzed their genetic characteristics for conservation. We genotyped 32 microsatellites and a mitochondrial D-loop region in 77 Misaki horses (80.2% of the population). The average number of alleles, observed heterozygosity, and expected heterozygosity were 3.4, 0.509, and 0.497, respectively. A neighbor-joining phylogenetic tree of individuals was constructed. Moreover, the results suggested that Misaki horses experienced a bottleneck, but it was neither severe nor recent. In addition, three mitochondrial haplotypes were confirmed. Consequently, we clarified the genetic background of Misaki horses that have been resident at Cape Toi for a long time.

Sexual precocity in male microminipigs evaluated immunohistologically using spermatogonial stem cell markers.

Microminipigs are one of the smallest miniature pigs characterized as sexually precocious; the males achieve sexual maturity at around 3-4.5 months of age. However, the physiology of this sexual precocity is still unclear. To understand sexual precocity in male microminipigs, we analyzed their testes at five developmental stages: neonatal (<7 days), 30-day-old, 45-day-old, 80-day-old, and adult (>24 months) stages. We used 4 pigs in each of the stages. To analyze testicular development histologically, the seminiferous tubule diameter (SD) was measured, and the presence or absence of the seminiferous lumen was confirmed. Changes in the expression of pluripotency markers, DBA, UCHL1, ZBTB16, and vimentin, were evaluated immunohistologically. For the analyses, cells positive for DBA, UCHL1, and ZBTB16 per 150 round seminiferous tubules in cross sections from each testis were counted to evaluate the total number of positive cells. The number of positive cells per 100 Sertoli cells (DBA+/Sertoli, UCHL1+/Sertoli, and ZBTB16+/Sertoli) was calculated to compare the five developmental stages. Histologically, SDs became larger with piglet growth, and precocity was confirmed; seminiferous lumens were observed from the 30-day-old stage. Immunohistologically, the number of DBA+/Sertoli, which indicates the number of gonocytes, decreased rapidly to an undetectable level by the 45-day-old stage. In the same period, the number of UCHL1+/Sertoli, which indicates total SSCs, increased significantly, suggesting that the proliferation of SSCs was accelerated before 30 days of age. Consequently, our study clarified that differentiation of SSCs in microminipigs started during the fetal period, the differentiation of gonocytes and proliferation of SSCs was then accelerated before 30 days of age, and the early phase of spermatogenesis was finally completed at around 45 days after birth. Consequently, sexual precocity in male microminipigs was characterized by a shorter duration of the early phase of spermatogenesis.

X monosomy in the endangered Kiso horse breed detected by a parentage test using sex chromosome linked genes and microsatellites.

A routine parentage test as part of a conservation program for Kiso horses identified a possible sex chromosome anomaly in a 7 months-old filly because of an aberrant result using LEX3, an X-linked marker. We then analyzed X-linked markers (LEX26, TKY38, and TKY270), Y-linked markers (Eca.YH12, Eca.YM2, Eca.YA16, and the sex-determining region Y gene), and an X/Y marker (Amelogenin gene). This analysis demonstrated that the filly had not inherited an X chromosome from her sire. A karyotyping analysis confirmed that the filly was 63,XO. As it was suspected that the horse would be sterile, we avoided using the horse as a broodmare; the information should also serve to prevent unnecessary conflict between owners transferring and receiving the horse.

Variation in the MC1R, ASIP, and MATP genes responsible for coat color in Kiso horse as determined by SNaPshot™ genotyping.

Kiso horse is a breed of Japanese native horses. In this study, to clarify coat color gene variation in Kiso horses, we used SNaPshot™ genotyping to evaluate variation in MC1R, ASIP, and MATP genes at the Extension (E), Agouti (A), and Cream dilution (C) loci. The coat color of 149 horses was documented. The coat color of 140, 3, and 6 horses was bay, chestnut, and buckskin, respectively. Furthermore, the frequency of alleles E, e, A, a, C, and Cr was 0.80, 0.20, 0.86, 0.14, 0.98, and 0.02, respectively. Current status of coat color genes in Kiso horses was clarified, and this information will help plan further conservation of the horses.

Response to estrus induction with abortion treatment in microminipigs on different days after insemination.

In microminipigs, estrus induction with abortion treatment, which is typically performed between 25 and 40 days after mating, is not always successful. Thus, the authors hypothesized that it may be more difficult to induce estrus by treating microminipigs approximately 40 days after mating. Accordingly, in this study, estrus induction was performed with abortion treatment in four microminipigs as follows: 0.3 mg of cloprostenol, a prostaglandin F2-alpha analog, was administered (day 0); after 24 h, 0.15 mg of cloprostenol and 250 IU of equine chorionic gonadotrophin were administered intramuscularly and simultaneously (day 1); after 96 h, 120 IU of human chorionic gonadotropin was injected intramuscularly (day 4). These treatments were compared at two different stages of pregnancy: early treatment (26.5 ± 0.7 days) and late treatment (38.3 ± 0.8 days). In the early treatment, all four microminipigs exhibited estrus on day 5, whereas in the late treatment, estrus was observed clearly in only two pigs on day 6 and slightly in 1 pig on day 10, whereas it was unclear in 1 pig. These results suggest that it is difficult to induce estrus with abortion treatment in microminipigs at approximately 40 days after mating.

Genetic relationship between Miyako and Yonaguni horses native to Okinawa based on polymorphisms of microsatellites.

The Miyako and Yonaguni horses are native horses in Okinawa. Here, we evaluated their genetic relationship using microsatellite data and Kiso horses, which have four subpopulations, as a reference population for evaluating this relationship. Microsatellite data from 35 Miyako, 78 Yonaguni, and 172 Kiso horses were evaluated using the STRUCTURE software for analyzing multilocus genotype data to investigate the population structures and their underlying relationship. The results of the STRUCTURE analysis were stable when ΔK was 2, suggesting that the Okinawan horses are different from the Kiso horses. Moreover, the results were also stable when ΔK was 6; the sample was then divided into four subpopulations of the Kiso horses and two Okinawan horse breeds. However, the diagrams from the STRUCTURE analysis were unstable when ΔK was 3. These results suggest that the genetic relationship of the Okinawan horse breeds may be close, similar to that among the subpopulations of the Kiso horses.

Accuracy of follicle count and ovulation confirmation using magnetic resonance imaging in microminipigs with normal estrus cycles.

Magnetic resonance imaging (MRI) is suggested to be useful for counting follicles and confirming ovulation in microminipigs. However, its accuracy is unknown. We have compared the number of follicles counted by MRI to that of corpus hemorrhagicum confirmed directly by visual inspection. The follicles of 17 microminipigs were counted by using ovarian MRI on a 0.4 Tesla MRI System every 24 hr after estrus until follicle images disappeared. Then, we performed laparotomy to count their corpus hemorrhagicum. Significant correlation was observed between follicle counts obtained using MRI (5.18 ± 1.78 per head) and the numbers of corpus hemorrhagicum (5.47 ± 1.74 per head). In conclusion, follicle counts using 0.4-T MRI were reliable, and confirmed microminipig ovulation.

Characterization of domestic pig spermatogenesis using spermatogonial stem cell markers in the early months of life.

In pigs, spermatogonial stem cells (SSCs), which include gonocytes and undifferentiated spermatogonia, are classically defined as being present till up to 2 months of life. However, knowledge about this transition from gonocytes to undifferentiated spermatogonia in pigs is limited. In this study, we characterized pig SSCs in neonatal (n = 6), one-month-old (1-mo-old, n = 6), two-month-old (2-mo-old, n = 6), and adult testes (n = 6) anatomically, histologically, and immunohistologically. Anatomically, testicular circumference (TC) was measured at each development stage. Histologically, the presence or absence of seminiferous lumen was confirmed, and seminiferous tubule diameter (SD) was measured. Immunohistologically, changes in expression of pluripotent markers: DBA, UCHL1, ZBTB16, and POU5F1, were evaluated. For the analyses, cells positive for DBA, ZBTB16, UCHL1, and Vimentin per 150 round seminiferous tubules of cross sections from each testis were counted to evaluate the total number of positive cells, and then the positive cells per 100 Sertoli cells (UCHL1+/Sertoli, DBA+/Sertoli, and ZBTB16+/Sertoli) were calculated to compare the four developmental stages. Anatomically, piglet testes became larger with increasing age. Histologically, there was no seminiferous lumen in piglet testes, and only a single layer of cells was observed within the seminiferous tubules. Immunohistologically, the average number of DBA+/Sertoli, which indicates the number of gonocytes, was significant lowerthan that of UCHL1+/Sertoli (P < 0.05), which indicates the number of total SSCs in neonatal testes, suggesting that spermatogenesis had already started at birth. In 2-mo-old testes, although the average number of UCHL1+/Sertoli was the same as that in neonatal and 1-mo-old testes, the average number of DBA+/Sertoli decreased rapidly to an undetectable level. Moreover, the numbers of ZBTB16+/Sertoli and DBA+/Sertoli in neonatal testes were similar, and the number of ZBTB16+/Sertoli in 1-mo-old testes was significantly lower than that of DBA+/Sertoli (P < 0.05). Since ZBTB16 may be a marker for stable gonocytes or spermatogonia, the multiplication of the SSCs in neonatal testes may not be very vigorous, and this may be accelerated around one month of age. Consequently, our study clarified that differentiation of pig SSCs starts during the fetal period; then, transition of SSCs from gonocytes to undifferentiated spermatogonia is accelerated at one month of age and finally completed at two months of age.