Quality and fertilizing ability of frozen-thawed porcine sperm separated using a migration sedimentation method.

We evaluated the quality and fertilizing ability of frozen-thawed porcine sperm that were selected using a commercially available device (MIGLIS, Menicon Life Science) consisting of three parts: an outer lid, an inner lid, and a tube. Firstly, to determine an adequate concentration of caffeine for separation, frozen-thawed sperm were incubated with different concentrations of caffeine (0, 1, 2.5, 5, and 10 mM) in a MIGLIS device. To determine the appropriate incubation time for separating sperm in the MIGLIS device, frozen-thawed sperm were incubated with 2.5 mM caffeine for 5, 10, 15, or 20 min. To evaluate the fertilization and embryo development of oocytes fertilized with frozen-thawed sperm separated into two regions (outer and inner) in the MIGLIS device, the separated sperm from the three boars was used to fertilize in vitro-matured oocytes and cultured in vitro for 7 days. Sperm quality parameters of sperm collected from the inner tube after incubation with 2.5 mM caffeine were superior to sperm incubated without caffeine. Moreover, sperm collected from the inner tube after incubation for 10 min had a higher progressive motility. The rate of blastocyst produced from spermatozoa collected from the inner tube after incubation with 2.5 mM caffeine for 10 min significantly increased compared to that produced from spermatozoa from the outer tube, regardless of the boar. In conclusion, sperm sorting using the MIGLIS device may be useful for separating high-quality sperm after incubation with 2.5 mM caffeine for 10 min to improve blastocyst formation.

Development of porcine embryos cultured in media irradiated with ultraviolet-C.

Sterilization of the culture medium using ultraviolet (UV)-C reduces the potential adverse effects of microorganisms and allows for long-term use. In the present study, we investigated the effects of a medium directly irradiated with UV-C prior to in vitro culture on the development and quality of porcine in vitro-fertilized embryos and the free amino acid composition of the culture media. The culture media (porcine zygote medium [PZM-5] and porcine blastocyst medium [PBM]) were irradiated with UV-C at 228 and 260 nm for 1 and 3 days, respectively. Next, the culture media were irradiated with UV-C at 228 nm for 3, 7, or 14 days. After in vitro fertilization, the embryos were cultured in the UV-C-irradiated media for 7 days. Free amino acid levels in culture media irradiated with 228 and 260 nm UV-C for 3 days were analysed. The blastocyst formation rate of embryos cultured in media irradiated with 260 nm UV-C for 3 days was significantly lower than that of embryos cultured in non-irradiated control media. However, 228 nm UV-C irradiation for up to 14 days did not affect blastocyst formation rates and quality in the resulting blastocysts. Moreover, 260 nm UV-C irradiation significantly increased the taurine concentration in both culture media and decreased methionine concentration in the PBM. In conclusion, UV-C irradiation at 228 nm before in vitro culture had no detrimental effects on embryonic development. However, 260 nm UV-C irradiation decreased embryo development and altered the composition of free amino acids in the medium.

Effects of ergothioneine supplementation on the quality of liquid-preserved and frozen-thawed boar semen.

This study examined the effects of ergothioneine (EGT) supplementation as an antioxidant on the quality of boar spermatozoa when using liquid and frozen preservation methods. In the first experiment, boar semen was preserved in an extender supplemented with 0, 50, 100 and 200 µM EGT, at 15 °C, part of the samples for one and another part for three weeks. In comparison with the control (without EGT), EGT supplementation at 100 µM significantly increased the percentage of total motility of spermatozoa that were preserved as a liquid both for one and three weeks (P < 0.05). EGT supplementation did not affect the quality of preserved spermatozoa, irrespective of the EGT concentration. In the second experiment, semen was frozen and thawed in the freezing extender supplemented with 0, 50, 100 and 200 µM EGT. In comparison with the control, the 100 µM EGT supplementation significantly increased the percentages of total and progressive motility of frozen-thawed spermatozoa (P < 0.05). EGT (100 µM) supplementation did not affect the viability, the plasma membrane integrity, or the acrosomal integrity of frozen-thawed spermatozoa. These findings indicate that supplementing extenders with 100 µM EGT may improve the motility of boar sperm in both liquid and freezing preservation methods.

Disruption of cell proliferation and apoptosis balance in the testes of crossbred cattle-yaks affects spermatogenic cell fate and sterility.

The balance between proliferation, differentiation and apoptosis is well-coordinated in spermatogenesis for the timely production of appropriate numbers of sperm in animals. Disruption or decrease in sperm production is due to many conditions, including changes in testicular cell fate balance. Interspecies hybridization of domestic yaks and cattle results in sterility in males because of spermatogenic arrest; however, the underlying mechanisms involved in sterility are still unclear. In the present study, we investigated the proliferation and apoptosis status during the development of yaks and crossbred cattle-yaks using immunohistochemistry of proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays. Testicular tissues from yaks (immature: 1 year old, mature: 2-3 years old) and backcrossed hybrids (2 year old) were collected and used to investigate the expression of each parameter in testicular cells. During the maturation of yak testes, proliferation and apoptosis became active only in spermatogenic cells, and not in other somatic cells, such as Sertoli cells, myoid cells and Leydig cells. Furthermore, hybrid cattle-yak testes maintained proliferation ability but less apoptotic ability in spermatogenic cells when compared to yaks of the same age, suggesting that normal spermatogenic cell fate control is disrupted by changes in the balance between proliferation and apoptosis. In addition, Leydig cell proliferation rate was higher than apoptosis rate in the cattle-yak testes, indicating an increased number of Leydig cells, which may affect spermatogenesis through changes in steroidogenesis. Although epigenetic changes may be involved in cattle-yak testes, further studies are needed to clarify the modulation of proliferation and apoptosis to elucidate the mechanisms of infertility in hybrid cattle-yak males.

Aberrant levels of DNA methylation and H3K9 acetylation in the testicular cells of crossbred cattle-yak showing infertility.

Although the interspecies hybridization of bovids, such as cattle-yak (Bos taurus × Bos grunniens), has heterosis benefits, the infertility of hybrid males affects the maintenance of dominant traits in subsequent generations. To achieve reproductive capacity, male germ cell development requires coordinated changes in gene expression, including DNA methylation and generalized histone modifications. Although gene expression-related mechanisms underlying hybrid male sterility have been investigated recently, information on the cell types and stage-specific controls remains limited. Here, we used immunohistochemistry and image analyses to evaluate the 5-methylcytosine (5MC) and acetyl-histone H3 Lys9 (AcK9) expression in all spermatogonia and testicular somatic cell types to determine their roles in cattle-yak spermatogenesis. Testicular tissues from yak (1-3 years old) and backcrossed hybrids (2 years old) were used. In yak, the AcK9 expression levels increased in all cell types during maturation, but the 5MC expression levels did not change until reaching 3 years when they increased in all testicular cell types, except spermatogonia. Cattle-yak hybrids showed higher 5MC expression levels and different AcK9 expression levels in all cell types compared to the same-aged yak. These results suggested that both gene modulation by AcK9 and constant levels of DNA methylation are required for spermatogenesis during maturation in yak. Therefore, inappropriate expression levels of both AcK9 and DNA methylation might be the major factors for disruption of normal germ cell development in cattle-yak. Additionally, various modulations occurred depending on the cell type. Further experiments are needed to identify the stage-specific gene expression modulations in each cell type in yak and cattle-yak to potentially solve the infertility issue in crossbreeding.

Abnormal functions of Leydig cells in crossbred cattle-yak showing infertility.

In Mongolia, yak (Bos grunniens) are able to live in alpine areas and their products greatly influence the lives of the local people. Increased vigour in hybridized yak and cattle can offer benefits for livestock farmers. However, male hybrids show reproductive defects resulting from spermatogenesis arrest, affecting the conservation and maintenance of dominant traits in the next generation. The underlying mechanisms involved in hybrid cattle-yak infertility have recently been investigated; however, the genetic cause is still unclear. Androgens and androgen receptor (AR) signalling are required for spermatogenesis. We, therefore, evaluated the expression of AR, 3ß-hydroxysteroid dehydrogenase (3ßHSD) and 5α-reductase 2 (SRD5A2) in Leydig cells to investigate their function in cattle-yak spermatogenesis. Testicular tissues from yaks (1-3 years old) and hybrids (F1-F3, 2 years old) were collected and subjected to immunohistochemistry and image analyses to investigate the expression of each parameter in the Leydig cells. After maturation at 2 years, the expression levels of AR increased and the levels of 3ßHSD decreased, but the SRD5A2 levels remained constant in yak. However, the cattle-yak hybrid F2 showed immature testicular development and significantly different expression levels of AR and 3ßHSD compared with mature yak. These results suggest that the decreased expression of AR and increased expression of 3ßHSD in the Leydig cells of cattle-yak hybrid testes may represent one of the causes of infertility. Our study might help in solving the problem of infertility in crossbreeding.

Sensitivity of the meiotic stage to hyperthermia during in vitro maturation of porcine oocytes.

The present study was conducted to clarify whether the meiotic stage of porcine oocytes has the highest sensitivity to hyperthermia during in vitro maturation by evaluating meiotic competence and DNA damage. Oocytes were exposed to 41 °C for 12 h at various intervals during 48 h of maturation culture. When the oocytes were exposed to 41 °C from 12 to 24 h of the maturation culture, the proportion of oocytes reaching metaphase II (MII) decreased as compared to the control oocytes cultured at 38.5 °C (P < 0.05). Moreover, the proportions of DNA fragmentation in all oocytes exposed to 41 °C in each culture period after 12 h from the start of maturation culture were significantly higher (P < 0.05) than for the control oocytes. When the meiotic stage of oocytes cultured at 38.5 °C between 12 and 24 h was examined, the majority of oocytes remained at the germinal vesicle (GV) stage at 12 h and approximately half of the oocytes reached metaphase I (MI) at 24 h. These results indicate that the meiotic stage of porcine oocytes having the highest sensitivity to hyperthermia during in vitro maturation is a transition period from the GV stage to the MI stage.

CHARACTERISTICS AND FERTILITY OF SUMATRAN TIGER SPERMATOZOA CRYOPRESERVED WITH DIFFERENT SUGARS.

BACKGROUND: Cryopreservation of semen is one of the most important methods for the preservation of endangered tigers. OBJECTIVE: This study evaluated the effects of sugar supplementation on the cryosurvival of spermatozoa from Sumatran tigers (Panthera tigris sumatrae). MATERIALS AND METHODS: The post-thaw characteristics and fertility of spermatozoa cryopreserved with different sugars (glucose, lactose, and trehalose) were evaluated using heterologous in-vitro fertilisation with cat oocytes. RESULTS: All parameters of post-thaw spermatozoa significantly decreased as compared with those of fresh spermatozoa. The index of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose. The percentage of total fertilisation for tiger spermatozoa cryopreserved with trehalose was significantly lower than that for control cat spermatozoa. CONCLUSION: Our findings indicated that supplementation with lactose or glycerol as the main sugar in the egg yolk extender resulted in a better motility and fertility potential for post-thawed spermatozoa.

Isolation of Japanese encephalitis virus and a novel insect-specific flavivirus from mosquitoes collected in a cowshed in Japan.

Cattle do not generally appear to develop severe viremia when infected with Japanese encephalitis virus (JEV), and they can be infected without showing clinical signs. However, two cattle in Japan recently died from JEV infection. In this study, we investigated the presence of different species of mosquitoes and flavivirus in a cowshed in the southwest region of Japan. In this cowshed, the two most common species of mosquitoes collected were Culex tritaeniorhynchus (including Culex pseudovishnui) and Anopheles sinensis. We performed virus isolation from the collected mosquitoes and obtained two flaviviruses: JEV and a novel insect-specific flavivirus, tentatively designated Yamadai flavivirus (YDFV). Phylogenetic analysis revealed that all three JEV isolates belonged to JEV genotype I and were closely related to a JEV strain that was isolated from the brains of cattle exhibiting neurological symptoms in Japan. Genetic characterization of YDFV revealed that the full genome RNA (10,863 nucleotides) showed homology with the Culex-associated insect-specific flaviviruses Quang Binh virus (79% identity) and Yunnan Culex flavivirus (78% identity), indicating that YDFV is a novel insect-specific flavivirus.

Formation of an Antral Follicle-Like Structure by Bovine Cumulus-Oocyte Complexes Embedded with Fragmin/Protamine Microparticles.

Fragmin/protamine microparticles (F/P MPs) approximately 0.5-1 µM in diameter were prepared by the simple mixing of fragmin with protamine. This study investigated the effects of F/P MP-containing collagen gels as a hormone carrier on the formation of antral follicle-like structures and on the development of growing bovine oocytes. The supplementation of F/P MPs in collagen gels contributed to the beneficial effects of follicle stimulating hormone (FSH) on the formation and size of antral follicle-like structures. The F/P MPs may serve as potential hormone carriers for the growth of cultured bovine oocytes from early antral follicles.

Effects of dibutyryl cyclic adenosine monophosphate and human chorionic gonadotropin on the formation of antral follicle-like structures by bovine cumulus-oocyte complexes.

This study evaluated the effect of dibutyryl cyclic adenosine monophos-phate (dbcAMP) and human chorionic gonadotropin (hCG) on the formation of antral follicle-like structures (AFLSs) and on the meiotic status of bovine cumulus- oocyte complexes (COCs) embedded in collagen gel. Supplementation with dbcAMP increased the mean diameter of AFLSs during days 4-8 of culture compared with that of control COCs, irrespective of the concentration of dbcAMP used (0.5-2.0 mM). When the embedded COCs were cultured for 8 days with hCG, the diameters of AFLSs after 4 days of culture tended to be lower in the supplemented COCs than in the control COCs without hCG, irrespective of the concentration used (1-100 IU/mL). Supplementation with 10 IU/mL hCG increased the concentrations of anti-Müllerian hormone but not progesterone and oestradiol in the culture medium after 4 days of culture. Almost all oocytes collected from AFLSs had resumed meiosis by the end of culture, irrespective of supplementation of dbcAMP and hCG. These results indicate that although dbcAMP had a positive effect on AFLS formation and development, supplementation with hCG was detrimental. Moreover, hCG supplementation did not influence the luteinisation of granulosa cells in the AFLS for 4 days after the start of culture.

Nuclear status and DNA fragmentation of oocytes from porcine, bovine and feline ovaries stored at 4 degrees C for 5 days.

BACKGROUND: The cooling of mammalian oocytes to sub-physiological temperatures is widely known to affect their viability through the induction of various abnormalities at all stages of meiosis. OBJECTIVE: This study was to compare the kinetics of nuclear status and oocyte damage in porcine, bovine and feline ovaries stored at 4 degrees C for 5 days. METHODS: The nuclear status and oocyte quality during storage were evaluated before and after maturation culture. RESULTS: The cold storage of ovaries decreased the proportions of porcine and bovine oocytes that remained at the germinal vesicle stage before maturation culture. The maturation rates of oocytes decreased with increasing storage time, independent of species. None of the porcine oocytes reached metaphase II (MII) after 1 day of storage. In contrast, bovine and feline oocytes from ovaries that were stored for 2 days and 3 days reached MII. DNA fragmentation in porcine oocytes from ovaries stored for 1 day was significantly higher than that in bovine and feline oocytes. CONCLUSION: The maturation competency of oocytes after the cold storage of ovaries could be related to the meiotic resumption of oocytes during storage and the occurrence of DNA fragmentation in oocytes during maturation culture.

Effects of skim-milk supplementation on the quality and penetrating ability of boar semen after long-term preservation at 15 °C.

This study investigated the effects of skim-milk supplementation on the quality and penetrating ability of boar semen preserved at 15 °C. When boar semen samples were preserved in Modified Modena extender supplemented with various concentrations (0, 7.5, 15, 30 and 50 mg/mL) of skim milk powder at 15 °C for 4 weeks, higher sperm motility and viability were observed in the case of 7.5 mg/mL skim-milk supplementation compared with the control group (0 mg/mL) during the preservation (P < 0.05). When in vitro matured oocytes were co-incubated with boar sperm that had been preserved in Modified Modena extender with three different concentrations (0, 7.5 or 15 mg/mL) of skim milk powder at 15 °C for two weeks, there were no apparent effects of skim-milk supplementation on the rates of fertilisation and development to blastocysts of oocytes after co-incubation. However, the monospermic fertilisation rate of sperm preserved with 15 mg/mL skim milk powder was higher (P < 0.05) than that of fresh non-preserved sperm, but did not differ among the preservation groups. The results indicate that the supplementation of Modified Modena extender with 7.5 mg/mL skim milk powder improves the motility and viability, but not the penetrating ability, of sperm after liquid preservation for at least two weeks.

Cell cycle analysis and interspecies nuclear transfer of cat cells treated with chemical inhibitors.

This study investigated the effect of chemical inhibitors on the cell-cycle synchronisation in cat fibroblast cells and evaluated the development of interspecies embryos reconstructed from cat donor cells and enucleated bovine oocytes. Cat fibroblast cells were treated with 15 µg/mL roscovitine or 0.05 µg/mL deme-colcine prior to cell cycle analysis and nuclear transfer. The percentage of cat fibroblast cells arrested at the G0/G1 phase in the roscovitine group was similar to that in the control group without any treatment. The percentage of cells arrested at the G2/M phase was significantly higher in the demecolcine group than in the control group. The fusion rate of interspecies couplets was significantly greater in the roscovitine group than in the control group. Most embryos stopped the development at the 2- or 4-cell stage, and none developed into blastocysts. Chemical inhibitor-induced donor cell cycle synchronisation did not overcome developmental arrest in interspecies cloned embryos.

Selection of spermatozoa with high motility and quality from bovine frozen-thawed semen using the centrifuge-free device.

This study aimed to assess the potential of the centrifuge-free commercial device (MIGLIS®) in selecting functional frozen-thawed bovine sperm by migration-sedimentation, its effect on embryo development, and compare the potential with that of centrifugation-based techniques, including washing and Percoll density gradient centrifugation (DGC). In experiment 1, different dilutions (1.5×, 2×, and 3×) of frozen-thawed spermatozoa were assessed to identify the adequate one for the MIGLIS method. In experiment 2, the recovery rates, quality, and reactive oxygen species (ROS) concentrations of the spermatozoa selected using MIGLIS, washing, and Percoll DGC were compared. In experiment 3, the resultant in vitro fertilised embryos from spermatozoa selected using the three methods were evaluated for blastocyst formation rates and intracellular ROS concentrations at the 2-4 cell stage. The intracellular ROS concentrations were investigated using 2', 7'-dichlorodihydrofluorescein diacetate staining. Using the MIGLIS device, significantly more spermatozoa were recovered at 2× dilution compared with the other dilution ratio, but the motility was not affected by the dilution ratio. On the selection of spermatozoa using the three methods, employing MIGLIS decreased the recovery rates. However, the MIGLIS method increased motility, viability, and acrosome integrity rates compared to those in spermatozoa from the other methods. The ROS concentration of spermatozoa in the MIGLIS method was significantly lower than that in the washing method. Nevertheless, blastocyst formation rates were similar among the three methods, but the ROS concentration of early-stage embryos produced using MIGLIS was significantly lower than those produced using Percoll DGC. In conclusion, the MIGLIS method has the potential to select functional, high-quality frozen-thawed bovine spermatozoa.

Novel Approach for Evaluating Pregnancy-Associated Glycoprotein and Inflammation Markers during the Postpartum Period in Holstein Friesian Cows.

We evaluated the relationship between decreased pregnancy-associated glycoprotein (PAG) levels, inflammatory parameters (serum amyloid A [SAA] and milk amyloid A [MAA]), postpartum inflammatory conditions (mastitis, ketosis, and follicular cysts), and the FOXP3 gene. Nineteen Holstein-Friesian cows were included in this study. Up to approximately eight weeks after delivery, weekly health examinations were performed for mastitis and ketosis, and reproductive organ ultrasonography was performed. The decreasing PAG rate was negatively correlated with SAA concentration (r = -0.493, p = 0.032). Cows with mastitis exhibited a slower trend of PAG decrease (p = 0.095), and a greater percentage of these cows had MAA concentrations above 12 µg/mL (p = 0.074) compared with those without mastitis. A negative correlation, although nonsignificant (r = -0.263, p = 0.385), was observed between the day-open period and decreased PAG rate. The day-open period was correlated with the presence or absence of follicular cysts (p = 0.046). Four cows that developed follicular cysts were homozygous for the G allele of the FOXP3 gene related to repeat breeders. These results indicate a relationship between a decreased PAG rate and inflammatory status during the postpartum period. Thus, suppressing inflammation during the perinatal period may improve reproductive efficiency in the dairy industry.

Cryopreservation for bovine embryos in serum-free freezing medium containing silk protein sericin.

Because the use of serum in the embryo cryopreservation increases the probability of animal health problems such as bovine spongiform encephalopathy (BSE) and viral infections, this study was conducted to examine the effects of sericin supplementation for serum-free freezing medium on the survival and development of bovine embryos after freezing-thawing and direct transfer to recipients. When in vitro-produced bovine embryos were frozen conventionally in the freezing medium supplemented with various concentrations (0.1%, 0.5%, and 1.0%) of sericin, the percentages of damaged zona pellucida, survival, and development of frozen-thawed embryos were similar to those of embryos frozen in freezing medium supplemented with 0.4% bovine serum albumin (BSA) and 20% fetal bovine serum (FBS) (0.4BSA/20F; control). When in vivo-derived embryos were frozen with 0.4BSA/20F (control), 0.5% sericin +20% FBS (0.5S/20F) or 0.5% sericin (0.5S) and were subsequently transferred directly to recipients, the percentages of recipients with pregnancy and normal calving in the 0.5S/20F group were higher than those in the control group (47.3% vs. 40.1% and 94.6% vs. 87.3%, respectively). Moreover, the percentages of recipients with pregnancy and normal calving (42.2% and 92.4%, respectively) in the 0.5S group were similar with those of other groups. In conclusion, these results indicate that serum-free freezing medium supplemented with sericin is available for the cryopreservation of bovine embryos and that it is beneficial for the elimination of a potential source of biological contamination by serum or BSA.

Comparison of activation ability between feline and bovine oocytes.

Research comparing the activation sensitivity of oocytes to chemical treatment among mammalian species remains limited. We compared the activation ability of oocytes from bovine and feline ovaries when treated with ethanol alone, with ethanol and cycloheximide, and without any chemical treatment; the oocytes were then cultured for 72 h. After in vitro maturation (IVM), 5% of feline oocytes were activated and 1% were cleaved, whereas there were no prematurely activated bovine oocytes. Activation rates with ethanol and ethanol/cycloheximide were significantly higher (P < 0.01) in bovine oocytes than in feline oocytes (74.2% vs. 34.1% and 86.3% vs. 52.5%, respectively). Thus, our findings indicate that feline oocytes can be prematurely activated by the end of IVM, and that bovine oocytes may have a higher sensitivity of parthenogenetic activation to chemical treatment than do feline oocytes.

Practical Application of a Urinary Zearalenone Monitoring System for Feed Hygiene Management of a Japanese Black Cattle Breeding Herd-Relevance to Anti-Müllerian Hormone and Serum Amyloid A Clarified from a Two-Year Survey.

In this study, a herd of Japanese Black (JB) breeding cattle with sporadic reproductive disorders was continuously monitored for an additional year to assess the effects of the urinary zearalenone (ZEN) concentration and changes in parameters (AMH and SAA) with time-lag variables and herd fertility (reproductive performance). This herd had high (exceeded the Japanese dietary feed regulations) urinary ZEN and rice straw ZEN concentrations (1.34 mg/kg). Long-term data of the herd with positive ZEN exposure revealed a decreasing ZEN concentration in urine and a gradual decrease in the AMH level with age. The AMH level was significantly affected by the ZEN value 2 months earlier and the AMH level in the previous month. The changes in ZEN and SAA values were significantly affected by the ZEN and SAA values in the previous month. Additionally, calving interval data between pre-monitoring and post-monitoring showed a significantly different pattern. Furthermore, the calving interval became significantly shorter between the time of contamination (2019) and the end of the monitoring period (2022). In conclusion, the urinary ZEN monitoring system may be a valuable practical tool for screening and detecting herd contamination in the field, and acute and/or chronic ZEN contamination in dietary feeds may affect herd productivity and the fertility of breeding cows.