Severe body condition loss lowers hepatic output of IGF1 with adverse effects on the dominant follicle in dairy cows.

The severe loss of body condition score (BCS) during the early lactation period has been associated with infertility in cows. However, the mechanisms are not fully understood. The aim of this study was to examine the effect of BCS loss on liver health, and ovarian functions in cows during early lactation. Retrospectively multiparous cows from two farms were categorized based on units of BCS (1-5 scale) loss as Moderate (MOD, <0.75 units; n = 11) or Severe (SEV, ≥0.75 units; n = 9) loss groups. From Weeks -3 to 7, relative to calving, MOD and SEV cows lost on average 0.4 and 1.0-unit BCS, respectively. All data except hepatic transcriptomes were analyzed with PROC MIXED procedure of SAS. The plasma concentration of non-esterified fatty acids at Week 0 and 1, ß-hydroxy butyrate at Week 1, and γ-glutamyl transferase at Weeks 1 and 7 relative to calving were higher in SEV cows. Hepatic transcriptome analysis showed that 1 186 genes were differentially expressed in SEV (n = 3) compared to MOD (n = 3) cows at Week 7 after calving. Pathway analysis revealed that significant DEGs in SEV cows enriched in lipid metabolisms including, lipid metabolic process, ether lipid metabolism, fatty acid beta-oxidation, fatty acid biosynthetic process, fatty acid metabolic process, fat digestion and absorption, linoleic acid metabolism, alpha-linolenic acid metabolism. The impaired liver function in SEV cows was associated with 1.5-fold reduction of hepatic IGF1 gene expression and lower serum IGF1 concentrations. At the ovarian level, SEV cows had lower IGF1 concentration in the follicular fluid of the dominant follicle of the synchronized follicular wave compared to that of MOD cows at 7 weeks after calving. Further, the follicular fluid concentration of estradiol-17ß was lower in SEV cows along with lower transcript abundance of genes from granulosa cells associated with dominant follicle competence, including CYP19A1, NR5A2, IGF1, and LHCGR. These data show that SEV loss of BCS during early lactation leading up to the planned start of breeding is associated with liver dysfunction, including lower IGF1 secretion, and impaired function of the dominant follicle in the ovary.

Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos.

CRISPR/Cas9 technology is a powerful tool used for genome manipulation in different cell types and species. However, as with all new technologies, it still requires improvements. Different factors can affect CRISPR/Cas efficiency in zygotes, which influence the total cost and complexity for creating large-animal models for research. This study evaluated the importance of zygote cell cycle stage between early-injection (within 6 h post activation/fertilization) versus late-injection (14-16 h post activation/fertilization) when the CRISPR/Cas9 components were injected and the inhibition of the homologous recombination (HR) pathway of DNA repair on gene editing, embryo survival and development on embryos produced by fertilization, sperm injection, somatic cell nuclear transfer, and parthenogenetic activation technologies. Injections at the late cell cycle stage decreased embryo survival (measured as the proportion of unlysed embryos) and blastocyst formation (68.2%; 19.3%) compared to early-stage injection (86.3%; 28.8%). However, gene editing was higher in blastocysts from late-(73.8%) vs. early-(63.8%) injected zygotes. Inhibition of the HR repair pathway increased gene editing efficiency by 15.6% in blastocysts from early-injected zygotes without compromising embryo development. Our finding shows that injection at the early cell cycle stage along with HR inhibition improves both zygote viability and gene editing rate in pig blastocysts.

Tauroursodeoxycholic acid/TGR5 signaling promotes survival and early development of glucose-stressed porcine embryos†.

Conditions of impaired energy and nutrient homeostasis, such as diabetes and obesity, are associated with infertility. Hyperglycemia increases endoplasmic reticulum stress as well as oxidative stress and reduces embryo development and quality. Oxidative stress also causes deoxyribonucleic acid damage, which impairs embryo quality and development. The natural bile acid tauroursodeoxycholic acid reduces endoplasmic reticulum stress and rescues developmentally incompetent late-cleaving embryos, as well as embryos subjected to nuclear stress, suggesting the endoplasmic reticulum stress response, or unfolded protein response, and the genome damage response are linked. Tauroursodeoxycholic acid acts via the Takeda-G-protein-receptor-5 to alleviate nuclear stress in embryos. To evaluate the role of tauroursodeoxycholic acid/Takeda-G-protein-receptor-5 signaling in embryo unfolded protein response, we used a model of glucose-induced endoplasmic reticulum stress. Embryo development was impaired by direct injection of tauroursodeoxycholic acid into parthenogenetically activated oocytes, whereas it was improved when tauroursodeoxycholic acid was added to the culture medium. Attenuation of the Takeda-G-protein-receptor-5 precluded the positive effect of tauroursodeoxycholic acid supplementation on development of parthenogenetically activated and fertilized embryos cultured under standard conditions and parthenogenetically activated embryos cultured with excess glucose. Moreover, attenuation of tauroursodeoxycholic acid/Takeda-G-protein-receptor-5 signaling induced endoplasmic reticulum stress, oxidative stress and cell survival genes, but decreased expression of pluripotency genes in parthenogenetically activated embryos cultured under excess glucose conditions. These data suggest that Takeda-G-protein-receptor-5 signaling pathways link the unfolded protein response and genome damage response. Furthermore, this study identifies Takeda-G-protein-receptor-5 signaling as a potential target for mitigating fertility issues caused by nutrient excess-associated blastomere stress and embryo death.

Histone Lysine Demethylases KDM5B and KDM5C Modulate Genome Activation and Stability in Porcine Embryos.

The lysine demethylases KDM5B and KDM5C are highly, but transiently, expressed in porcine embryos around the genome activation stage. Attenuation of KDM5B and KDM5C mRNA hampered embryo development to the blastocyst stage in fertilized, parthenogenetically activated and nuclear transfer embryos. While KDM5B attenuation increased H3K4me2-3 levels on D3 embryos and H3K4me1-2-3 on D5 embryos, KDM5C attenuation increased H3K9me1 on D3 embryos, and H3K9me1 and H3K4me1 on D5 embryos. The relative mRNA abundance of EIF1AX and EIF2A on D3 embryos, and the proportion of D4 embryos presenting a fluorescent signal for uridine incorporation were severely reduced in both KDM5B- and KDM5C-attenuated compared to control embryos, which indicate a delay in the initiation of the embryo transcriptional activity. Moreover, KDM5B and KDM5C attenuation affected DNA damage response and increased DNA double-strand breaks (DSBs), and decreased development of UV-irradiated embryos. Findings from this study revealed that both KDM5B and KDM5C are important regulators of early development in porcine embryos as their attenuation altered H3K4 and H3K9 methylation patterns, perturbed embryo genome activation, and decreased DNA damage repair capacity.

Tauroursodeoxycholic acid acts via TGR5 receptor to facilitate DNA damage repair and improve early porcine embryo development.

DNA damage associated with assisted reproductive technologies is an important factor affecting gamete fertility and embryo development. Activation of the TGR5 receptor by tauroursodeoxycholic acid (TUDCA) has been shown to reduce endoplasmic reticulum (ER) stress in embryos; however, its effect on genome damage responses (GDR) activation to facilitate DNA damage repair has not been examined. This study aimed to investigate the effect of TUDCA on DNA damage repair and embryo development. In a porcine model of ultraviolet light (UV)-induced nuclear stress, TUDCA reduced DNA damage and ER stress in developing embryos, as measured by γH2AX and glucose-regulated protein 78 immunofluorescence, respectively. TUDCA was equally able to rescue early embryo development. No difference in total cell number, DNA damage, or percentage of apoptotic cells, measured by cleaved caspase 3 immunofluorescence, was noted in embryos that reached the blastocyst stage. Interestingly, Dicer-substrate short interfering RNA-mediated disruption of TGR5 signaling abrogated the beneficial effects of TUDCA on UV-treated embryos. Quantitative PCR analysis revealed activation of the GDR, through increased messenger RNA abundance of DNAPK, 53BP1, and DNA ligase IV, as well as the ER stress response, through increased spliced XBP1 and X-linked inhibitor of apoptosis. Results from this study demonstrated that TUDCA activates TGR5-mediated signaling to reduce DNA damage and improve embryo development after UV exposure.

Granulosa cells of prepubertal cattle respond to gonadotropin signaling and upregulate genes that promote follicular growth and prevent cell apoptosis.

Oocytes collected from prepubertal animals are known to be less developmentally competent than those from adult animals. There is evidence suggesting that acquisition of developmental competence in bovine oocytes may be linked to the expression profile of genes in the granulosa cells (GCs). Cumulus-oocyte complexes (COC) and GCs were collected from 12 Holstein heifers between 2 and 6 months of age (nine follicle-stimulating hormone [FSH] treated and three untreated) and eight FSH-treated cows. The COCs from prepubertal animals were matured, fertilized, and cultured in vitro to assess development to the blastocyst stage. The relative messenger RNA (mRNA) abundance of FSHR, StAR, CYP19A1, HSD3B1, CX43, FOXO1, and XIAP in GCs were quantified by real-time quantitative polymerase chain reaction. Results from this study revealed that GCs of prepubertal animals respond to FSH treatment by increasing mRNA levels of genes promoting estradiol synthesis and follicular growth ( FSHR and CYP19A1), and preventing cell apoptosis ( XIAP), and by decreasing mRNA levels of genes promoting progesterone production ( StAR and HSD3B1). This study also revealed that the relative mRNA abundance of FOXO1 in GCs is associated with oocyte competence to support embryo development to the blastocyst stage in prepubertal Holstein heifers.

Interval of gonadotropin administration for in vitro embryo production from oocytes collected from Holstein calves between 2 and 6 months of age by repeated laparoscopy.

Laparoscopic Ovum Pick-Up (LOPU) in calves followed by in vitro embryo production (IVEP) and transfer (ET) into adult recipients has great potential for accelerated genetic gain through shortening of the generation interval. In this study, 11 Holstein calves were subjected to up to six LOPU procedures between the ages of 2-6 months at 2-3 weeks interval. In all cases, the animals received a CIDR 5 days prior to LOPU and were gonadotropin-stimulated starting at 72 h before LOPU using one of three protocols that were rotated twice among the animals during the study. Calves were injected with FSH every 12 h (FSH12h), or every 8 h (FSH8h) or every 8 h until -36 h from LOPU at which point the FSH was replaced with a single dose of 400 IU eCG (FSH8h-eCG). No statistical differences were observed among the 3 treatments in terms of mean follicles available for aspiration (35.7 ±â€¯16 vs. 38.5 ±â€¯25 vs. 31.1 ±â€¯22), mean oocytes recovered (26.5 ±â€¯14 vs. 21.6 ±â€¯10 vs. 19.4 ±â€¯14) and cleavage rate (66.0 ±â€¯14 vs. 61.1 ±â€¯11 vs. 72.2 ±â€¯8), for FSH12h, FSH8h and FSH8h-eCG, respectively. However, FSH8h-eCG resulted in a significantly higher rate of transferable embryos (17.5 ±â€¯8%) compared with FSH12h (8.9 ±â€¯5%, P < 0.05). Oocytes from follicles of ≥5 mm in diameter yielded a higher rate (P < 0.05) of development to the blastocyst stage (13.8%) than those collected from <5 mm follicles (6.8%). Animal age, by comparing animals at <100, 101 to 130 and > 130 days of age, did not affect the mean number of follicles (34.2 ±â€¯15 vs. 39.3 ±â€¯26 vs. 31.6 ±â€¯25), the mean number of oocytes recovered (21.2 ±â€¯10 vs. 24.5 ±â€¯15 vs. 22.6 ±â€¯17), and the cleavage rate (68.6 ±â€¯11 vs. 61.7 ±â€¯12 vs. 70.7 ±â€¯10%), respectively. However, animals in the older age range had significantly higher development to the blastocyst stage (19.9 ±â€¯6 vs. 9.5 ±â€¯8%, P < 0.01) and better embryo quality, as evidenced by higher average cell numbers (119.1 ±â€¯47 vs. 91.5 ±â€¯25, P < 0.05) compared with those in the lower age. Finally, we tested the benefits of relieving endoplasmic reticulum stress by supplementing the culture medium with 50 µM tauroursodeoxycholic acid (TUDCA) and found a numerically higher rate of development to the blastocyst stage (21.1 ±â€¯8 vs. 18.6 ±â€¯4%), but not statistically different, compared with control culture. Overall, our findings indicate that a significant number of transferable embryos (range 10-30) can be produced from Holstein calves before they reach 6 months of age.

Double-strand DNA breaks are mainly repaired by the homologous recombination pathway in early developing swine embryos.

DNA double-strand breaks (DSBs) are less frequent than single-strand breaks but have more harmful consequences on cell survival and physiology. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are the two main pathways that are responsible for DSB repair in eukaryotic cells, but their importance for the preservation of genome stability in totipotent blastomeres of early developing embryos has not been determined. In this study, we observed that the chemical inhibition of HR or both pathways, but not NHEJ alone, increased the number of DSBs, reduced embryo development to the blastocyst stage, and resulted in embryos with higher proportions of apoptotic cells. Targeted knockdown of ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related; HR regulators) and DNA-dependent protein kinase (NHEJ regulator) mRNAs revealed that the attenuation of HR or both HR and NHEJ regulators severely impaired blastocyst formation and quality. Attenuation of ATM alone resulted in a higher incidence of DSBs, lower development and embryo quality, and increased mRNA abundance of genes that are involved in either repair pathway. These findings indicate that HR is the main pathway responsible for the promotion of DSB repair in early developing embryos, and that ATM seems to be more important than ATR in the regulation of the HR pathway in mammalian embryos.-Bohrer, R. C., Dicks, N., Gutierrez, K., Duggavathi, R., Bordignon, V. Double-strand DNA breaks are mainly repaired by the homologous recombination pathway in early developing swine embryos.

Relief of endoplasmic reticulum stress enhances DNA damage repair and improves development of pre-implantation embryos.

Early-cleaving embryos are known to have better capacity to reach the blastocyst stage and produce better quality embryos compared to late-cleaving embryos. To investigate the significance of endoplasmic reticulum (ER) stress on early embryo cleavage kinetics and development, porcine embryos produced in vitro were separated into early- and late-cleaving groups and then cultured in the absence or presence of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Developing embryos were collected at days 3 to 7 of culture for assessment of ER stress status, incidence of DNA double-strand breaks (DSBs), development and total cell number. In the absence of TUDCA treatment, late-cleaving embryos exhibited ER stress, higher incidence of DNA DSBs, as well as reductions in development to the blastocyst stage and total embryo cell numbers. Treatment of late-cleaving embryos with TUDCA mitigated these effects and markedly improved embryo quality and development. These results demonstrate the importance of stress coping responses in early developing embryos, and that reduction of ER stress is a potential means to improve embryo quality and developmental competence.

The effect of age and length of gonadotropin stimulation on the in vitro embryo development of Holstein calf oocytes.

The use of oocytes recovered from prepubertal donors for in vitro embryo production has great potential for accelerating the rate of genetic gain in the dairy industry. However, these oocytes are known to be less developmentally competent than those from adult donors. In this study, we investigated the effect of age and gonadotropin stimulation in Holstein heifers subjected to oocyte collection every two weeks between 2 and 6 months of age. In order to assess the effect of gonadotropin stimulation, animals were subjected to one of three treatments, namely Short (ST; 36-42 h), Long (LT; ≥72 h) and No Treatment (NT) prior to laparoscopic ovum pick-up (LOPU). Our results show that the LT significantly improved the proportion of large follicles (>5 mm diameter) present in the ovary (LT 34.0% vs. ST 11.2% vs. NT 2.4%, P < 0.05), as well as the percentage of good-quality cumulus oocyte complexes (COCs) recovered (LT 95.3 ± 18% vs. ST 85.4 ± 22% vs. NT 82.2 ± 14%, P < 0.05) and blastocyst rate (LT 36.7 ± 26% vs. ST 18.3 ± 15% vs. NT 16.7 ± 9%, P < 0.05). Recovery rate was affected by treatment (LT 70.4 ± 25 vs. ST 85.4 ± 29 vs. NT 72.7 ± 23, P < 0.05). To assess the impact of age, data was grouped into <100 days (A), 100-130 days (B) and >130 days (C) of age at LOPU. We found that as animals got older, although the average number of COCs per donor per LOPU declined (A: 17.5 ± 11 vs. B: 14.7 ± 7 vs. C: 11.9 ± 8), the blastocyst rate increased (A: 12.8 ± 20% vs. B: 17.1 ± 21% vs. C: 21.8 ± 25%, P < 0.05). We also evaluated the incidence of polyspermy and confirmed it is a critical limitation for IVF in calf oocytes. The incidence of polyspermy was unaffected by gonadotropin treatment, but significantly decreased with age. The capacity for full development to term of in vitro produced embryos from calf oocytes was tested by embryo transfer into 21 synchronized adult recipients, which resulted in 13 pregnancies (62%), full development to term and healthy calves born. Finally, the study allowed evaluating the safety of the procedure since, on average, each animal was subjected to 8 LOPU procedures over a period of 4 months. Our results showed that the procedure is safe (no incidents during laparoscopy), and was not harmful for the reproductive future of the animals, as those that were bred became pregnant after reaching sexual maturity.

Efficacy of the porcine species in biomedical research.

Since domestication, pigs have been used extensively in agriculture and kept as companion animals. More recently they have been used in biomedical research, given they share many physiological and anatomical similarities with humans. Recent technological advances in assisted reproduction, somatic cell cloning, stem cell culture, genome editing, and transgenesis now enable the creation of unique porcine models of human diseases. Here, we highlight the potential applications and advantages of using pigs, particularly minipigs, as indispensable large animal models in fundamental and clinical research, including the development of therapeutics for inherited and chronic disorders, and cancers.

Endoplasmic reticulum stress, genome damage, and cancer.

Endoplasmic reticulum (ER) stress has been linked to many diseases, including cancer. A large body of work has focused on the activation of the ER stress response in cancer cells to facilitate their survival and tumor growth; however, there are some studies suggesting that the ER stress response can also mitigate cancer progression. Despite these contradictions, it is clear that the ER stress response is closely associated with cancer biology. The ER stress response classically encompasses activation of three separate pathways, which are collectively categorized the unfolded protein response (UPR). The UPR has been extensively studied in various cancers and appears to confer a selective advantage to tumor cells to facilitate their enhanced growth and resistance to anti-cancer agents. It has also been shown that ER stress induces chromatin changes, which can also facilitate cell survival. Chromatin remodeling has been linked with many cancers through repression of tumor suppressor and apoptosis genes. Interplay between the classic UPR and genome damage repair mechanisms may have important implications in the transformation process of normal cells into cancer cells.

The use of an angularis oris axial pattern flap in a dog after resection of a multilobular osteochondroma of the hard palate.

An 11-year-old neutered male boxer was presented for treatment of a multilobular osteochondroma of the hard palate. The mass was surgically resected and the hard palate defect was reconstructed using an angularis oris axial pattern buccal mucosal flap. No local recurrence was reported 6 mo after surgery.