RNA polymerase II pausing is essential during spermatogenesis for appropriate gene expression and completion of meiosis.

Male germ cell development requires precise regulation of gene activity in a cell-type and stage-specific manner, with perturbations in gene expression during spermatogenesis associated with infertility. Here, we use steady-state, nascent and single-cell RNA sequencing strategies to comprehensively characterize gene expression across male germ cell populations, to dissect the mechanisms of gene control and provide new insights towards therapy. We discover a requirement for pausing of RNA Polymerase II (Pol II) at the earliest stages of sperm differentiation to establish the landscape of gene activity across development. Accordingly, genetic knockout of the Pol II pause-inducing factor NELF in immature germ cells blocks differentiation to spermatids. Further, we uncover unanticipated roles for Pol II pausing in the regulation of meiosis during spermatogenesis, with the presence of paused Pol II associated with double-strand break (DSB) formation, and disruption of meiotic gene expression and DSB repair in germ cells lacking NELF.

Use of Transabdominal Ultrasound and Maternal Hormone Testing for the Prenatal Monitoring of Equine Fetal Enlarged Bladder.

Ultrasound examination during late gestation is one of the best methods for monitoring potential pregnancy risks. Enlarged bladder is a urological disorder rarely observed in equine fetuses. This clinical case report aimed to present a case illustrating the development of equine fetal enlarged bladder using transabdominal ultrasound examinations and maternal hormone evaluation during gestation. An 8-year-old Hokkaido native pony was impregnated by embryo transfer, and at 215 days of gestation, abnormalities of the fetal bladder were detected. The bladder volume increased with gestational age, and a second bladder was observed at 257 days of gestation. No abnormalities were observed in the fetal kidneys. Moreover, the maternal plasma progesterone concentration was measured throughout the gestation period. The progesterone concentration was elevated from 36 weeks of gestation until parturition. At 363 days of gestation, parturition induction was conducted, and a foal successfully delivered. This case report is the first to describe the development of equine fetal enlarged bladder and record the corresponding ultrasound and hormone profiles.

First Kiso pony foal produced via transfer of long-distance shipped fresh embryo to Hokkaido native pony.

Japanese native horses, which consists of 8 breeds, are threatened with extinction. Embryo transfer (ET) is used to reproduce endangered animals in various mammalian species. We aimed to perform ET using native ponies from Kiso and Hokkaido as donors and recipients, respectively. ET operation included long-distance transport of non-cryopreserved embryos from Nagano Prefecture to Hokkaido. Embryos were transported 1500 km over 9 h in a container maintained at 22°C. After transferring two embryos to two recipients, one mare delivered a healthy live foal. These results demonstrated that reciprocal ET with long-distance transportation of fresh embryos between the isolated breeds may allow for the proliferation of Japanese native horses.

Ultrasonographic examination of equine fetal growth parameters throughout gestation in pony for Equine-Assisted Therapy.

Equine-Assisted Therapy (EAT) is gaining popularity. Ultrasound examination is used to decrease the abortion rate in horses. In this study, to monitor fetal well-being throughout the gestation for EAT, we measured fetal heart rate (FHR), fetal eye orbit (FEO), fetal gonad length (FGL), fetal kidney length (FKL), and the combined thickness of the uterus and placenta (CTUP) by ultrasonography in pony mares. Additionally, we measured the plasma progesterone (P4) and estradiol (E2) concentrations in pregnant horses using enzyme immunoassay. The FGL peaked at week 32 and then decreased to term, and a strong correlation (r=0.72, P<0.001) between the FGL and E2 concentration was observed. A strong correlation with gestational age was detected among the FEO (r=0.96, P<0.001), FKL (r=0.85, P<0.001), and CTUP (r=0.96, P<0.001). The P4 concentration peaked at week 10, decreased to low levels (below 5 ng/ml), and peaked before parturition. In conclusion, this study provides information on fetal growth throughout gestation in pony mares for EAT. In addition, it revealed the relationship between ultrasonographic profile and plasma hormone concentrations during gestation.

Evaluating the use of piezo manipulator, laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos.

The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non-surgical transcervical procedure and divided into three groups based on their size (A ≤ 300 µm, 300 µm300 µm). However, more research is required to find the best method for embryos ≥700 µm.

Reduction of seminal plasma concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa.

This study was conducted to investigate the effect of different levels of seminal plasma (SP) and cold-shock on ram spermatozoa during 36 h storage at 5°C. In both ejaculated spermatozoa coated with egg yolk (second ejaculate; coated spermatozoa) and epididymal spermatozoa, samples were treated with 0, 50 and 100% seminal plasma. Different levels of seminal plasma were added on the basis of ram spermatocrit (32%). Then half of aliquots were suddenly put on ice water (cold-shock) and other half were gradually (0.25°C/min) chilled (non- cold shock). Sperm motility, viability and functional membrane integrity were determined in both aliquots at 0, 12, 24 and 36 h storage at 5°C. Under non- cold shock and cold-shock conditions, coated spermatozoa treated with 0% SP showed the highest motility compared to ejaculated spermatozoa (first ejaculate; uncoated spermatozoa) after 12, 24 and 36 h of storage at 5°C (P<0.05). Under non- cold shock and cold-shock conditions, viability and functional membrane integrity was higher in the coated spermatozoa treated with 0% SP than in the uncoated spermatozoa during 36 h storage (P<0.05). There was no significant difference between coated spermatozoa treated with 0 and 50% SP in the percentage of motility and viability after 24 and 36 h of storage (P>0.05). Under non- cold shock and cold-shock conditions, the percentage of motility of epididymal spermatozoa treated with 0% SP was significantly (P<0.05) higher than those treated with 100% SP after 36 h of storage at 5°C. In conclusion, removal of seminal plasma and/or reduction (up to 50%) of its concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa.

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.

Polymyxin B enhances acrosomal exocytosis triggered by calcium and the calcium ionophore A23187 in ejaculated boar spermatozoa.

Polymyxin B (PMB) is beneficial for boar semen storage since it neutralizes the endotoxin of bacteria. However, the direct effect of PMB on boar spermatozoa has been unknown. This study aimed to examine the effect of PMB on acrosomal exocytosis, an essential process for successful fertilization in boar spermatozoa. Ejaculated spermatozoa stored with BTS extender at 17°C were washed and incubated with 0-100 µM PMB for 20 min and then examined for % total motililty, vigor grade and viability. None of the parameters was significantly different between 0 and 50 µM PMB with a gradual decline at higher concentrations. Thus the effect on acrosomal exocytosis was investigated at 0-50 µM of PMB. Spermatozoa were preincubated with PMB for 10 min, incubated for stimulation of acrosomal exocytosis with Ca2+ and the calcium ionophore A23187 and then fixed with glutaraldehyde at 5, 10 and 15 min. Preincubation with PMB at 0.01-50 µM and 0.05-50 µM resulted in significant enhancement of acrosomal exocytosis at 10 min and 15 min of incubation, respectively. Preincubation with PMB followed by incubation without A23187 did not affect acrosomal exocytosis. These results suggest that PMB exerts effects on the acrosomal exocytosis triggered by Ca2+ and A23187 in boar spermatozoa.

Methodological improvement of fluorescein isothiocyanate peanut agglutinin (FITC-PNA) acrosomal integrity staining for frozen-thawed Japanese Black bull spermatozoa.

This study aimed to improve the staining of frozen-thawed Japanese Black bull sperm acrosomes with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Spermatozoa were washed, fixed with 1-3% paraformaldehyde (PFA) in suspension for 10, 20, and 30 min, permeabilized with 0-2% Triton X-100 for 5 min, stained with FITC-PNA, and mounted with different antifade agents (0.22 M 1,4-diazabicyclo [2,2,2] octane (DABCO), SlowFade®, and ProLong®) in suspension (In-suspension) or on a smear (On-smear). The spermatozoa were categorized into seven pattern types either immediately or after storage for 24 hr. Experiment 1 showed that 1) the In-suspension method was better than the On-smear method; 2) if spermatozoa were stained using the In-suspension method and examined immediately, the best antifade agent was SlowFade®; 3) if samples were to be stored after staining using the On-smear method, DABCO should be avoided; 4) if spermatozoa were stained using the In-suspension method, storage of the stained samples was not recommended; and 5) if samples were to be stored after staining using the In-suspension method, ProLong® might be the best antifade agent. The results of experiment 2 showed that the concentration of Triton X-100 could be reduced to 0.1 from 1%. The results of experiment 3 showed that the paraformaldehyde concentration used for a 30 min fixation could be reduced from 3 to 2%. It is expected that the improved staining protocol will be useful to determine bull sperm acrosomal integrity.