Bulk vitrification of in vitro produced bovine zygotes without reducing developmental competence to the blastocyst stage.

Cryopreservation of mammalian zygotes can be advantageous since it enables their flexile use in time and space for alternative purposes such as genome editing. Here we report a simple, quick and inexpensive vitrification protocol for in vitro produced bovine zygotes which enables their bulk preservation. Slaughterhouse-derived oocytes were subjected to in vitro maturation and fertilization (IVF). Ten h after IVF, cumulus-enclosed zygotes were equilibrated in 2% (v/v) ethylene glycol + 2% (v/v) propylene glycol for 13-15 min then vitrified in groups of 52-100 in 2 µL microdrops of 17.5% (v/v) ethylene glycol + 17.5% (v/v) propylene glycol supplemented with 0.3 M sucrose and 50 mg/mL polyvinylpyrrolidone. The presence of cumulus cells is important for the success of the process. Therefore, we applied a modified IVF protocol using a short (30 min) co-incubation interval which allowed zygote culture with attached cumulus cells until vitrification and even reduced polyspermy rates without affecting the total fertilization rate. Vitrified zygotes were similar to their non-vitrified counterparts in terms of survival, post-warming development to the blastocyst stage and blastocyst quality measured by cell numbers and cryo-survival. In conclusion, our vitrification protocol integrated with the modified IVF system enabled the quick cryopreservation of bovine zygotes in large groups without reducing their developmental competence to the blastocyst stage.

Effects of reduced glutathione supplementation in semen freezing extender on frozen-thawed bull semen and in vitro fertilization.

During cryopreservation, spermatozoa may suffer cold and cryo-induced injuries -associated with alterations in cell defense systems- that are detrimental to their function and subsequent fertility. This study aimed to determine the efficacy of supplementing the semen freezing extender with the antioxidant reduced glutathione (GSH) in cattle. Semen was collected from four bulls and diluted in a freezing extender supplemented with or without GSH (0, 1, 5, and 10 mM) before the cooling step of the cryopreservation process. After thawing, the quality of the frozen-thawed semen was investigated for motility, viability, acrosomal and DNA integrity, and subsequent embryo development after in vitro fertilization of bovine oocytes. Additionally, semen from one of the bulls was used to analyze semen antioxidative potential, sperm penetration into oocytes, male pronucleus formation rate, and embryo DNA integrity. The sperm quality varied among bulls after GSH supplementation. One bull had decreased sperm total motility, and two bulls had decreased sperm DNA integrity. GSH supplementation had positive effects on embryo development for three bulls. Two of them showed both improved cleavage and blastocyst formation rates, while the other one only showed an improved cleavage rate. We observed positive effects on early male pronucleus formation and no negative effects on DNA integrity and cell number in blastocyst stage embryos. Although the effect varies depending on individual bulls and GSH concentration, GSH supplementation in semen may improve in vitro embryo production from frozen semen.

Differentially methylated CpG sites related to fertility in Japanese Black bull spermatozoa: epigenetic biomarker candidates to predict sire conception rate.

For semen suppliers, predicting the low fertility of service bull candidates before artificial insemination would help prevent economic loss; however, predicting bull fertility through in vitro assessment of semen is yet to be established. In the present study, we focused on the methylated CpG sites of sperm nuclear DNA and examined methylation levels to screen new biomarkers for predicting bull fertility. In frozen-thawed semen samples collected from Japanese Black bulls, for which the sire conception rate (SCR) was recorded, the methylation level of each CpG site was analyzed using human methylation microarray. According to regression analysis, 143 CpG sites related to SCR were significantly differentially methylated. Whole genome bisulfite sequence data were obtained from three semen samples and the differentially methylated regions (DMRs) that included the target CpG sites selected by human methylation microarray were confirmed. Using combined bisulfite restriction analysis, fertility-related methylation changes were detected in 10 DMRs. With the exception of one DMR, the methylation levels of these DMRs were significantly different between groups with high fertility (> 50%) and low fertility (< 40%). From multiple regression analysis of methylation levels and SCR, three DMRs were selected that could effectively predict bull fertility. We suggest that these fertility-related differences in spermatozoal methylation levels could be new epigenetic biomarkers for predicting bull fertility.

In vitro survival kinetics of microfluidic-sorted bovine spermatozoa.

BACKGROUND: The isolation and characterization of sperm subpopulations that can achieve fertilization is a major challenge of assisted reproduction methods. We focused on the microfluidic sperm sorter as a novel tool for collecting highly motile spermatozoa from heterogeneous semen samples. OBJECTIVES: This study primarily aims to obtain baseline information on sorted spermatozoa according to its characteristics and in vitro life span. MATERIALS AND METHODS: Frozen-thawed bull semen was subjected to microfluidic sperm sorting using diffuser-type microfluidic sperm sorter (DMSS). After sorting, samples were collected as the sorted spermatozoa and unsorted residual spermatozoa and incubated at 37°C for subsequent evaluation. The samples were assessed at different time points (0 or 1, 6, and 24 h) in terms of motility, which was measured by computer-assisted sperm analysis (CASA), membrane integrity, mitochondrial function, and adenosine triphosphate (ATP) production after sorting (0 h). To determine the characteristics and efficiency of DMSS sorting, the sorted spermatozoa were compared with samples collected using the swim-up method, a conventional method in motile sperm selection. RESULTS: A comparison between the sorted and residual spermatozoa demonstrated significantly higher motility parameters, membrane integrity, and mitochondrial function of the sorted spermatozoa until 6 h after incubation. The time course decrement of membrane and mitochondrial status were subjected to curve fitting and theoretically supported. Sperm ATP production measured immediately after sorting showed higher ATP generation of the sorted spermatozoa compared with the unsorted, frozen-thawed spermatozoa. The motility parameters and mitochondrial activity of DMSS-sorted spermatozoa were higher than the swim-up-collected spermatozoa (p < 0.05). DISCUSSION AND CONCLUSION: These results indicate that DMSS sorting can strictly select highly motile spermatozoa with the ability to maintain its membrane integrity and mitochondrial function related to ATP production. We speculate that the device that is able to sort high-quality spermatozoa can have great potential in assisted reproduction.

Bovine sperm selection procedure prior to cryopreservation for improvement of post-thawed semen quality and fertility.

BACKGROUND: The application of cryopreservation and artificial insemination technology have contributed to the advancement of animal reproduction. However, a substantial proportion of spermatozoa undergoes alterations and loses their fertility during cryopreservation, rendering the frozen-thawed semen impractical for routine use. Cryopreservation is known to reduce sperm lifespan and fertility. Variation in cryosurvival of spermatozoa from different sires and even with the individual sire is common in artificial insemination (AI) centers. Our goal is to improve post-thawed semen quality by optimization of cryopreservation technique through sperm selection prior to cryopreservation process. RESULTS: Our strategy of sperm selection based on rheotaxis and thermotaxis (SSRT) on macrosale in a rotating fluid flow demonstrated the ability to maintain the original pre-freezing structural integrity, viability and biological function related to fertilization competence. This strategy has a positive effect on the cryosurvival and fertilizing abilities of spermatozoa as supported by the improvement on pregnancy rate of Japanese Black heifers and Holstein repeat breeders. This technique protected further sublethal damage to bovine spermatozoa (higher % cryosurvival than the control) and resulted in the improvement of DNA integrity. Prefreeze selected spermatozoa demonstrated slower and controlled capacitation than unprocessed control which is thought to be related to sperm longevity and consequently to appropriate timing during in vivo fertilization. CONCLUSIONS: These results provide solid evidence that improvement of post-thawed semen quality by SSRT method is beneficial in terms of cryosurvival, longevity of post-thawed sperm, and optimization of in vivo fertilization, embryo development and calving as supported by the favorable results of field fertility study.

Live births from artificial insemination of microfluidic-sorted bovine spermatozoa characterized by trajectories correlated with fertility.

Selection of functional spermatozoa plays a crucial role in assisted reproduction. Passage of spermatozoa through the female reproductive tract requires progressive motility to locate the oocyte. This preferential ability to reach the fertilization site confers fertility advantage to spermatozoa. Current routine sperm selection techniques are inadequate and fail to provide conclusive evidence on the sperm characteristics that may affect fertilization. We therefore developed a selection strategy for functional and progressively motile bovine spermatozoa with high DNA integrity based on the ability to cross laminar flow streamlines in a diffuser-type microfluidic sperm sorter (DMSS). The fluid dynamics, with respect to microchannel geometry and design, are relevant in the propulsion of spermatozoa and, consequently, ultrahigh-throughput sorting. Sorted spermatozoa were assessed for kinematic parameters, acrosome reaction, mitochondrial membrane potential, and DNA integrity. Kinematic and trajectory patterns were used to identify fertility-related subpopulations: the rapid, straighter, progressive, nonsinuous pattern (PN) and the transitional, sinuous pattern (TS). In contrast to the conventional notion that the fertilizing spermatozoon is always vigorously motile and more linear, our results demonstrate that sinuous patterns are associated with fertility and correspond to truly functional spermatozoa as supported by more live births produced from predominant TS than PN subpopulation in the inseminate. Our findings ascertain the true practical application significance of microfluidic sorting of functional sperm characterized by sinuous trajectories that can serve as a behavioral sperm phenotype marker for fertility potential. More broadly, we foresee the clinical application of this sorting technology to assisted reproduction in humans.

Duodenal infusion of fatty acids differentially affects plasma glucagon-like peptide-1 and ghrelin concentrations in sheep.

The aim of this study was to investigate how intraduodenal infusions of fatty acids (FA) affect appetite-related gut peptides such as glucagon-like peptide-1 (GLP-1) and ghrelin in sheep. We hypothesized that these peptides can be highly reactive to unsaturated long-chain FA, because they are well known to decrease dry matter intake (DMI). Four ewes were fitted with a duodenal cannula and a jugular vein catheter for a 6-h duodenal infusion of the 9 FA (C8:0, C10:0, C12:0, C14:0, C16:0, C18:0, C18:1, C18:2, and C18:3) and water (control). The concentration of each FA was 1.6 g per metabolic body weight (BW), approximately corresponding to the amount of supplemented fat in a standard dairy cow diet. Each infusion was separated by at least 2 d. During the infusion period, blood samples were collected periodically to determine changes in plasma GLP-1, ghrelin, and metabolite concentrations. Duodenal infusions of C18:1, C18:2, and C18:3 led to higher plasma GLP-1 (P < 0.05) and lower glucose (P < 0.05) than control. Plasma ghrelin concentrations were greater in C18:1 and C18:3 infusions than control (P < 0.05). Plasma ketone bodies were higher in C8:0 and C10:0 infusions (P < 0.05), but plasma triglyceride concentrations were lower in C8:0, C10:0, C12:0, and C16:0 infusions (P < 0.05) than control. Fatty acid infusions except for C18:3 led to higher plasma NEFA concentrations than control (P < 0.05). These results confirmed that the hypophagic effect of dietary unsaturated long-chain FA is mediated by GLP-1 (an anorexigenic effect) secretion. However, we also observed higher plasma ghrelin (an orexigenic effect) partially by unsaturated long-chain FA. Thus, the gut peptide secretions when ruminant animals ingest FA supplements would complexly affect satiety and further studies are needed to determine their each impact on DMI.

Effects of insulin-like growth factor-1 on the in vitro maturation of canine oocytes.

The maturation rate of canine oocytes during in vitro maturation (IVM) needs to be improved. The present study was designed to evaluate the effects of insulin-like growth factor-1 (IGF-1) on the IVM of canine oocytes. Ovaries were obtained by ovariohysterectomy and were sliced to release cumulus-oocyte complexes (COCs). In Experiment 1, the effects of different concentrations of IGF-1 on the nuclear maturation of oocytes was investigated. The COCs were cultured in a modified medium (mTCM199) with IGF-1 (0, 0.5, 5, 10, and 50 µg/ml). At the end of the 48 h culture, oocytes were fixed and stained to evaluate their nuclear stage. Supplementation with 50 µg/ml IGF-1 induced a significantly higher metaphase II (MII) rate (P < 0.05) compared to the 0 and 0.5 µg/ml IGF-1 groups. In Experiment 2, the expression levels of insulin receptor (INSR), IGF-1 receptor (IGF-1R), and IGF-2 receptor (IGF-2R) genes, localized to canine oocytes and cumulus cells, were investigated before and after IVM. The expression level of IGF-1R in cumulus cells after IVM was higher than that before IVM (P < 0.05). In Experiment 3, it was investigated whether an inhibitor of PTEN (phosphatase and tensin homolog), bpV, affects the nuclear maturation of oocytes. Regardless of bpV supplementation at a concentration of 0.2 to 200 µmol/l, there was no significant difference in the proportion of oocytes that reached the MII stage. These results indicated that IGF-1 has a favorable effect on the IVM of canine oocytes, possibly through the stimulation of the Ras/MAPK pathway via IGF-1R expressed in cumulus cells.

Histochemical properties of bovine and ovine mammary glands during fetal development.

In order to obtain more information on the development of bovine and ovine fetal mammary glands, a series of mammary glands from fetuses of different ages were analyzed. A total of 16 bovine fetuses with curved crown rump lengths ranging from 12 cm (80 days) to 75 cm (240 days) and 15 ovine fetuses ranging from 55 days to 131 days were examined. We used hematoxylin and eosin stain and Oil-Red-O stain to analyze the developmental and morphogenetic processes of mammary glands. In addition, we used immunohistochemical staining to determine the pattern of expression of cytokeratin 18 (CK18) during luminal epithelial differentiation, α-smooth-muscle actin (α-SMA) for myoepithelial differentiation, Ki-67 for cell proliferation, and estrogen receptor α (ERα). Our analyzes showed: (a) The primary mammary duct begin to proliferate in a lengthwise within the teat at 90 days in bovine fetuses and 63 days in ovine fetus; (b) luminal epithelial cells and myoepithelial cells appeared from 90 days in bovine fetuses and 63 days in ovine fetus; (c) proliferation of epithelial cells appeared to coincide with the development of the primary and secondary ducts; and (d) ERα was not found in the fetal mammary gland, but adipocytes showed the presence of ERα. Overall, these results indicate that the sequence of events in the prenatal development of the mammary gland of sheep is similar to that of cattle.

Glutathione supplementation to semen extender improves the quality of frozen-thawed canine spermatozoa for transcervical insemination.

The present study was conducted to evaluate whether supplementation of semen extender with glutathione (GSH) can maintain the quality of frozen-thawed canine spermatozoa. Eighteen ejaculates were obtained from 5 dogs and placed in extender (20% egg yolk, Tris, citric acid, lactose, raffinose, antibiotics and 6.5% glycerol) containing 0 (control), 2.5, 5, 7.5 or 10 mM GSH. The samples were cooled to 4 C and then frozen in liquid nitrogen vapor. Motility parameters of the sperm were evaluated at 0, 1, 2, 3, 4, 12 and 24 h after thawing. Sperm motility was higher in the 5 mM GSH group than in the control or 2.5 and 10 mM GSH groups; this effect was observed at 1 to 24 h after thawing (P < 0.05). The 5 mM GSH group had a higher sperm viability index at 12 and 24 h after thawing compared with the other groups (P < 0.05). Acrosome integrity, evaluated at 4 h after thawing, was greater in two of the GSH-treated groups (5 and 10 mM) compared with the control. Lipid peroxidation (LP) levels immediately after thawing were lower in the 5 and 10 mM GSH groups compared with the control, while those at 12 h after thawing did not differ significantly. Frozen-thawed semen in the 5 mM GSH group was used for transcervical insemination of 4 bitches, resulting in delivery of 5 puppies from 2 bitches. These results indicate that supplementation of semen extender with 5 mM GSH was effective in improving motility, longevity and acrosomal integrity and inhibiting LP levels in post-thaw canine spermatozoa, without any adverse impacts on full-term development after transcervical insemination.

The developmental potential of oocytes is impaired in cattle with liver abnormalities.

Here, we investigated the effect of liver abnormality on the developmental potential of bovine oocytes. Good quality oocytes from healthy cows and from animals with a liver abnormality were matured and fertilized in vitro and then cultured to the blastocyst stage. On day 7 after fertilization, embryo cleavage and development were assessed. The concentrations of glucose, nonesterified fatty acid (NEFA), γ-glutamyl transpeptidase (γ-GTP), ß-hydroxybutyrate (BHBA) and glutathione were measured in follicular fluids (FF). The proportion of good quality oocytes and the frequency of development to the blastocyst stage were lower in the liver anomaly group than those of the control group (P<0.05). The concentrations of γ-GTP and BHBA in the FF of the liver anomaly group were higher than those of the control group (P<0.05). The concentration of glutathione in the FF of the liver anomaly group was lower than that of the control group (P<0.05). Moreover, there was a negative correlation between these concentrations and the proportions of oocytes that developed to the blastocyst stage (P<0.05). Supplementation of the culture medium with γ-GTP or BHBA did not affect the rate of oocyte maturation but did cause a concentration-dependent reduction in the frequency of fertilized oocytes that developed to the blastocyst stage. Our findings indicate that the quality of oocytes and their potential for development are lower in cattle with liver disorders than those in healthy cattle; one possible cause may be the high concentration of γ-GTP and/or BHBA in their FF.