The pFSH dose affects the efficiency of in vivo embryo production in Santa Inês ewes.

This study compared the follicular growth, superovulatory response, and in vivo embryo production after administering two doses of porcine follicle-stimulating hormone (pFSH) in Santa Inês ewes. The estrous cycle of 36 multiparous ewes was synchronized with the Day 0 protocol and superovulated with 133 mg (G133, n=18) or 200 mg (G200, n=18) of pFSH. Ultrasonographic evaluations of the ovaries were performed, ewes were mated and submitted to non-surgical embryo recovery. Viable blastocysts were stained with Nile Red and Hoechst. The G200 had a greater number of medium and large follicles, as well as a larger size of the third largest follicle. A total of 97.2% (35/36) of the ewes came into estrus and it was possible to transpose cervix in 80.6% (29/36). There were no effects of treatments in the response to superovulation, the proportion of ewes in which was possible to transpose the cervix, the number of corpora lutea, the number of anovulatory follicles, the proportion of ewes flushed with at least one recovered structure, number of recovered structures, number of viable embryos, viability rate, and recovery rate. The G200 ewes were in estrus for a longer period of time than the G133 ewes (54.0 ± 4.5 h vs. 40.3 ± 3.6 h) and produced more freezable embryos (6.5 ± 1.6 vs. 2.3 ± 0.7) than G133. Both doses promoted an efficient superovulatory response and did not affect embryonic lipid accumulation. The dose of 200 mg of pFSH showed greater potential to increase the superovulatory response, as it increased follicular recruitment and the recovery of freezable embryos.

Ian Wilmut (1944-2023).

Embryologist who cloned Dolly the sheep.

The GnRH Antagonist Degarelix Suppresses Gonadotropin Secretion and Pituitary Sensitivity in Midgestation Sheep Fetuses.

The specific role of gonadotropin-releasing hormone (GnRH) on brain sexual differentiation remains unclear. To investigate whether gonadotropin and, in turn, testosterone (T) secretion is regulated by GnRH during the critical period for brain differentiation in sheep fetuses, we attempted to selectively suppress pituitary-testicular activation during midgestation with the long-acting GnRH antagonist degarelix. Fetuses received subcutaneous injections of the antagonist or vehicle on day 62 of gestation. After 2 to 3 weeks we examined consequences of the intervention on baseline and GnRH-stimulated plasma luteinizing hormone (LH) and T levels. In addition, we measured the effect of degarelix-treatment on messenger RNA (mRNA) expression for the pituitary gonadotropins and key gonadal steroidogenic enzymes. Baseline and GnRH-stimulated plasma LH levels were significantly suppressed in degarelix-treated male and female fetuses compared to control values. Similarly, T concentrations were suppressed in degarelix-treated males. The percentage of LHß-immunoreactive cells colocalizing c-fos was significantly reduced by degarelix treatment indicating that pituitary sensitivity was inhibited. Degarelix treatment also led to the significant suppression of mRNA expression coding for the pituitary gonadotropin subunits and for the gonadal enzymes involved in androgen synthesis. These findings demonstrate that pharmacologic inhibition of GnRH early in gestation results in suppression of LH secretion and deficits in the plasma T levels of male lamb fetuses. We conclude that GnRH signaling plays a pivotal role for regulating T exposure during the critical period of sheep gestation when the brain is masculinized. Thus, disturbance to gonadotropin secretion during this phase of gestation could have long-term consequence on adult sexual behaviors and fertility.

Analysis of Polycerate Mutants Reveals the Evolutionary Co-option of HOXD1 for Horn Patterning in Bovidae.

In the course of evolution, pecorans (i.e., higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as "headgear," which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.

Fetal sheep support the development of hematopoietic cells in vivo from human induced pluripotent stem cells.

We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)-derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs.

Reducing lung liquid volume in fetal lambs decreases ventricular constraint.

BACKGROUND: This study evaluated whether an increased left ventricular (LV) pump function accompanying reduction of lung liquid volume in fetal lambs was related to increased LV preload, augmented LV contractility, or both. METHODS: Eleven anesthetized preterm fetal lambs (gestation 128 ± 2 days) were instrumented with (1) an LV micromanometer-conductance catheter to obtain LV end-diastolic volume (EDV) and end-diastolic pressure (EDP), the maximal rate of rise of LV pressure (dP/dtmax), LV output, LV stroke work, and LV end-systolic elastance (Ees), a relatively load-independent measure of contractility; (2) an endotracheal tube to measure mean tracheal pressure and to reduce lung liquid volume. LV transmural pressure was calculated as LV EDP minus tracheal pressure. RESULTS: Reducing lung liquid volume by 16 ± 4 ml kg-1 (1) augmented LV output (by 16%, P = 0.001) and stroke work (29%, P < 0.001), (2) increased LV EDV (12%, P < 0.001), (3) increased LV transmural pressure (2.2 mmHg, P < 0.001), (4) did not change LV dP/dtmax normalized for EDV (P > 0.7), and (5) decreased LV Ees (20%, P < 0.01). CONCLUSION: These findings suggest a rise in LV pump function evident after reduction of lung liquid volume in fetal lambs was related to increased LV preload secondary to lessening of external LV constraint, without any associated rise in LV contractility. IMPACT: This study has shown that reducing the volume of liquid filling the fetal lungs lessens the degree of external constraint on the heart. This lesser constraint permits a rise in left ventricular dimensions and thus greater cardiac filling that leads to increased left ventricular pumping performance. This study has defined a mechanism whereby a reduction in lung liquid volume results in enhanced pumping performance of the fetal heart. These findings suggest that a reduction in lung liquid volume which occurs during the birth transition contributes to increases in left ventricular dimensions and pumping performance known to occur with birth.

Immortalization of primary sheep embryo kidney cells.

Sheep primary epithelial cells are short-lived in cell culture systems. For long-term in vitro studies, primary cells need to be immortalized. This study aims to establish and characterize T immortalized sheep embryo kidney cells (TISEKC). In this study, we used fetal lamb kidneys to derive primary cultures of epithelial cells. We subsequently immortalized these cells using the large T SV40 antigen to generate crude TISEKC and isolate TISEKC clones. Among numerous clones of immortalized cells, the selected TISEKC-5 maintained active division and cell growth over 20 passages but lacked expression of the oncogenic large T SV40 antigen. Morphologically, TISEKC-5 maintained their epithelial aspect similar to the parental primary epithelial cells. However, their growth properties showed quite different patterns. Crude TISEKC, as well as the clones of TISEKC proliferated highly in culture compared to the parental primary cells. In the early passages, immortalized cells showed heterogeneous polyploidy but in the late passages the karyotype of immortalized cells became progressively stable, identical to that of the primary cells, because the TISEKC-5 cell line has lost the large SV40 T antigen expression, this cell line is a valuable tool for veterinary sciences and biotechnological productions.

Interaction of apoptosis and pluripotency related transcripts for developmental potential of ovine embryos produced in vitro at different oxygen concentrations.

The present study in sheep model was to find out the interaction of apoptotic transcripts, that is, Bcl2, Bax, Casp3, PCNA and p53 and pluripotency related transcripts, that is, Sox2, Nanog and Oct4 in ovine embryos produced in vitro at different O2 concentrations (20% and 5% O2) to compare their developmental potential. Oxygen concentrations did not influence the maturation and cleavage rate but the percentage of morula and blastocysts was significantly more at 5% as compared to 20% O2. A significant upregulated expression of Bcl2 and PCNA genes and significantly downregulated expression of Casp3 and p53 were observed in the blastocysts at 5% than those at 20% O2. The expression of Bax was not influenced by the O2 concentration. Among the pluripotency related transcripts, the expression of Oct4 was significantly upregulated and the expression of Sox2 and Nanog was significantly downregulated in embryos at 5% than at 20% O2. The study concluded that the embryos produced in vitro at low O2 (5%) concentration regulate the expression of developmental genes related to apoptosis and pluripotency to improve the developmental potential of embryos as compared to high O2 (20%) concentration.

Effects of maternal periconceptional undernutrition in sheep on offspring glucose-insulin axis function into adulthood.

Maternal periconceptional undernutrition (PCUN) affected fetal pancreatic maturation in late gestation lambs and impaired glucose tolerance in 10-month-old sheep. To examine the importance of the timing of maternal undernutrition around conception, a further cohort was born to PCUN ewes [undernourished for 61 d before conception (PreC), 30 d after conception (PostC), or 61 d before until 30 d after conception (PrePostC)], or normally fed ewes (Control) (n = 15-20/group). We compared glucose tolerance, insulin secretion, and sensitivity at 36 months of age. We also examined protein expression of insulin signalling proteins in muscle from these animals and in muscle from a fetal cohort (132 d of gestation; n = 7-10/group). Adult PostC and PrePostC sheep had higher glucose area under the curve than Controls (P = 0.07 and P = 0.02, respectively), whereas PreC sheep were similar to Controls (P = 0.97). PostC and PrePostC had reduced first-phase insulin secretion compared with Control (P = 0.03 and P = 0.02, respectively). PreC was similar to Control (P = 0.12). Skeletal muscle SLC2A4 protein expression in PostC and PrePostC was increased 19%-58% in fetuses (P = 0.004), but decreased 39%-43% in adult sheep (P = 0.003) compared with Controls. Consistent with this, protein kinase C zeta (PKCζ) protein expression tended to be increased in fetal (P = 0.09) and reduced in adult (P = 0.07) offspring of all PCUN ewes compared with Controls. Maternal PCUN alters several aspects of offspring glucose homeostasis into adulthood. These findings suggest that maternal periconceptional nutrition has a lasting impact on metabolic homeostasis of the offspring.

Loss of ITGB3 in ovine conceptuses decreases conceptus expression of NOS3 and SPP1: implications for the developing placental vasculature†.

During the peri-implantation period of pregnancy in sheep, there is an initial period of loose apposition of the elongating conceptuses (embryos and associated placental membranes) to the endometrial luminal epithelium (LE) that is followed by adhesion of the conceptus trophectoderm to the endometrial LE for implantation. Integrins and maternal extracellular matrix (ECM) molecules are major contributors to stable adhesion at implantation, and the ß3 integrin subunit (ITGB3) is implicated in the adhesion cascade for implantation in several species including the sheep. We blocked mRNA translation for trophectoderm-expressed ITGB3 by infusing morpholino antisense oligonucleotides into the uterine lumen of pregnant ewes on Day 9 to assess effects on conceptus elongation, and on Day 16 to assess effects on early placental development in sheep. Results indicate that sheep conceptuses elongate and implant to the uterine wall in the absence of ITGB3 expression by the conceptuses; however, loss of ITGB3 in conceptuses decreased the growth of embryos to Day 24 of gestation, and decreased expression of secreted phosphoprotein 1 (SPP1) and nitric oxide synthase 3 (NOS3). Abundant SPP1 was localized around the blood vessels in the placental allantoic membrane in normal sheep pregnancies. We hypothesize that NOS3 and SPP1 positively influence the development of the vasculature within the allantois, and that decreased expression of NOS3 and SPP1, in response to knockdown of ITGB3 in conceptuses, alters development of the vasculature in the allantois required to transport nutrients from the endometrium to support growth and development of the embryo.

Effect of maternal nutrient restriction on expression of glucose transporters (SLC2A4 and SLC2A1) and insulin signaling in skeletal muscle of SGA and Non-SGA sheep fetuses.

Maternal nutrient restriction (NR) causes small for gestational age (SGA) offspring, which are at higher risk for accelerated postnatal growth and developing insulin resistance in adulthood. Skeletal muscle is essential for whole-body glucose metabolism, as 80% of insulin-mediated glucose uptake occurs in this tissue. Maternal NR can alter fetal skeletal muscle mass, expression of glucose transporters, insulin signaling, and myofiber type composition. It also leads to accumulation of intramuscular triglycerides (IMTG), which correlates to insulin resistance. Using a 50% NR treatment from gestational day (GD) 35 to GD 135 in sheep, we routinely observe a spectral phenotype of fetal weights within the NR group. Thus, we classified those fetuses into NR(Non-SGA; n = 11) and NR(SGA; n = 11). The control group (n = 12) received 100% of nutrient requirements throughout pregnancy. At GD 135, fetal plasma and gastrocnemius and soleus muscles were collected. In fetal plasma, total insulin was lower in NR(SGA) fetuses compared NR(Non-SGA) and control fetuses (P < 0.01), whereas total IGF-1 was lower in NR(SGA) fetuses compared with control fetuses (P < 0.05). Within gastrocnemius, protein expression of insulin receptor (INSRB; P < 0.05) and the glucose transporters, solute carrier family 2 member 1 and solute carrier family 2 member 4, was higher (P < 0.05) in NR(SGA) fetuses compared with NR(Non-SGA) fetuses; IGF-1 receptor protein was increased (P < 0.01) in NR(SGA) fetuses compared with control fetuses, and a lower (P < 0.01) proportion of type I myofibers (insulin sensitive and oxidative) was observed in SGA fetuses. For gastrocnemius muscle, the expression of lipoprotein lipase (LPL) messenger RNA (mRNA) was upregulated (P < 0.05) in both NR(SGA) and NR(Non-SGA) fetuses compared with control fetuses, whereas carnitine palmitoyltransferase 1B (CPT1B) mRNA was higher (P < 0.05) in NR(Non-SGA) fetuses compared with control fetuses, but there were no differences (P > 0.05) for protein levels of LPL or CPT1B. Within soleus, there were no differences (P > 0.05) for any characteristic except for the proportion of type I myofibers, which was lower (P < 0.05) in NR(SGA) fetuses compared with control fetuses. Accumulation of IMTG did not differ (P > 0.05) in gastrocnemius or soleus muscles. Collectively, the results indicate molecular differences between SGA and Non-SGA fetuses for most characteristics, suggesting that maternal NR induces a spectral phenotype for the metabolic programming of those fetuses.

In vivo embryo production and recovery in lacaune ewes after imposing a superovulation treatment regimen is related to pFSH dose.

This study was conducted to assess effects of different doses of pFSH on follicular recruitment, superovulatory response, ova/embryo recovery, and embryo yield in lactating ewes. Ewes (n = 24) had a superovulation treatment regimen imposed. All ewes were implanted with a progesterone intravaginal device for 9 d, and administered either 100 (G-100) or 200 (G-200) mg pFSH, proportioned into six doses administered at 12-h intervals, starting 60 h before device removal. At 7 days subsequent to progesterone device removal, there were non-surgical embryo recoveries (NSER) from ewes having three or more corpora lutea. At the time of the first pFSH injection, number of antral follicles were similar (P < 0.05) between ewes in the G-100 and G-200 group, however, there were more 3.1-4.0 mm follicles in ewes of the G-200 than G-100 group at the time of the second pFSH administration. Estrous response and CL number were less (P < 0.05) in ewes of the G-100 (66.7 % and 2.6 ±â€¯0.7) than G-200 (91.7 % and 11.6 ±â€¯1.2) group. There were embryo collections from 100 % and 90.9 % of ewes in the G-100 and G-200 groups, respectively (P > 0.05). Viable embryo numbers and ova/embryo recovery rate were greater (P < 0.05) in ewes of the G-200 (6.9 ±â€¯1.1 and 67.8 %) than G-100 (1.0 ±â€¯0.5 and 27.6 %) group. A dose of 200 mg pFSH was more effective in inducing a superovulatory response and embryo yield after NSER in ewes, however, the 100 mg dose was insufficient for these purposes.

Leucine acutely potentiates glucose-stimulated insulin secretion in fetal sheep.

A 9-day infusion of leucine into fetal sheep potentiates fetal glucose-stimulated insulin secretion (GSIS). However, there were accompanying pancreatic structural changes that included a larger proportion of ß-cells and increased vascularity. Whether leucine can acutely potentiate fetal GSIS in vivo before these structural changes develop is unknown. The mechanisms by which leucine acutely potentiates GSIS in adult islets and insulin-secreting cell lines are well known. These mechanisms involve leucine metabolism, including leucine oxidation. However, it is not clear if leucine-stimulated metabolic pathways are active in fetal islets. We hypothesized that leucine would acutely potentiate GSIS in fetal sheep and that isolated fetal islets are capable of oxidizing leucine. We also hypothesized that leucine would stimulate other metabolic pathways associated with insulin secretion. In pregnant sheep we tested in vivo GSIS with and without an acute leucine infusion. In isolated fetal sheep islets, we measured leucine oxidation with a [1-14C] l-leucine tracer. We also measured concentrations of other amino acids, glucose, and analytes associated with cellular metabolism following incubation of fetal islets with leucine. In vivo, a leucine infusion resulted in glucose-stimulated insulin concentrations that were over 50% higher than controls (P < 0.05). Isolated fetal islets oxidized leucine. Leucine supplementation of isolated fetal islets also resulted in significant activation of metabolic pathways involving leucine and other amino acids. In summary, acute leucine supplementation potentiates fetal GSIS in vivo, likely through pathways related to the oxidation of leucine and catabolism of other amino acids.

On-farm lambing outcomes after transfer of vitrified and slow frozen embryos.

The aim of the present study was to determine the most suitable embryonic stage and embryo freezing technique for commercial implementation of frozen embryo trading by small-scale sheep producers. There was a 2 × 2 factorial design utilized for conducting the study consisting of two embryo stages (2-8 cells or morula/blastocyst) and two cryopreservation protocols (vitrification or slow-freezing). For the in vivo produced embryos, there were treatments of crossbred donor ewes to induce superovulation. Embryos were recovered surgically on either Day 2 or 5.5 after estrous onset. The embryos were cryopreserved using either a vitrification or slow-freezing method before there was transfer to recipients. Ovarian response, embryo survival and lambing outcomes were analyzed. There were no differences in number of recovered and fertilized embryos at the two embryonic developmental stages. There were no effects of embryonic stages and cryopreservation methods on pregnancy rate, twinning rate, fetal birth weights and lamb weight at 1 month of age. When there was use of vitrified embryos for transfers, there was a greater lamb weight at 2 months of age (8.38 ± 0.20 compared with 7.78 ± 0.21 kg; P = 0.044) than when there was transfer of embryos cryopreserved using slow freezing procedures. Considering economic and practical benefits to small-scale sheep farms, morula/blastocyst stage-embryo collection and transfer into the uterus is more efficacious than transferring 2-8 cells embryos into the oviduct. Results of this study may contribute to the genetic improvement in the flocks of small-scale sheep producers.

Sexing of pre-implantation ovine embryos through polymerase chain reaction-based amplification of GAPDH, SRY and AMEL genes.

The ability to identify the sex of embryo and control of sex ratio has a great commercial importance to livestock industry. Prediction of embryonic sex could be useful in the management decisions of sex selection in breeding programs. Several methods have been attempted to determine the sex but the polymerase chain reaction (PCR)-based sexing method is generally favoured, as it is cost effective, simple and reliable. The aim of the present study was to identify sex of sheep embryos produced in vitro through amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sex-determining region Y (SRY) and amelogenin genes present in genomic DNA (gDNA) of embryos through PCR. To avoid false interpretation of the result by no amplification of SRY in female embryos, a duplex PCR was approached to amplify combinedly SRY and GAPDH genes. Sex-specific blood was used in PCR as positive control. In vitro sheep embryos were produced as per standardized protocol of laboratory. Sexing of sex-specific blood and in vitro produced embryos were approached though PCR to amplify the respective genes using gDNA present in the sample without its traditional isolation. The accuracy of sex prediction for embryos was 100% by this procedure.

The Effect of Size, Maturation, Global Asphyxia, Cerebral Ischemia, and Therapeutic Hypothermia on the Pharmacokinetics of High-Dose Recombinant Erythropoietin in Fetal Sheep.

High-dose human recombinant erythropoietin (rEPO) is a promising potential neuroprotective treatment in preterm and full-term neonates with hypoxic-ischemic encephalopathy (HIE). There are limited data on the pharmacokinetics of high-dose rEPO in neonates. We examined the effects of body weight, gestation age, global asphyxia, cerebral ischemia, hypothermia and exogenous rEPO on the pharmacokinetics of high-dose rEPO in fetal sheep. Near-term fetal sheep on gestation day 129 (0.87 gestation) (full term 147 days) received sham-ischemia (n = 5) or cerebral ischemia for 30 min followed by treatment with vehicle (n = 4), rEPO (n = 8) or combined treatment with rEPO and hypothermia (n = 8). Preterm fetal sheep on gestation day 104 (0.7 gestation) received sham-asphyxia (n = 1) or complete umbilical cord occlusion for 25 min followed by i.v. infusion of vehicle (n = 8) or rEPO (n = 27) treatment. rEPO was given as a loading bolus, followed by a prolonged continuous infusion for 66 to 71.5 h in preterm and near-term fetuses. A further group of preterm fetal sheep received repeated bolus injections of rEPO (n = 8). The plasma concentrations of rEPO were best described by a pharmacokinetic model that included first-order and mixed-order elimination with linear maturation of elimination with gestation age. There were no detectable effects of therapeutic hypothermia, cerebral ischemia, global asphyxia or exogenous treatment on rEPO pharmacokinetics. The increase in rEPO elimination with gestation age suggests that to maintain target exposure levels during prolonged treatment, the dose of rEPO may have to be adjusted to match the increase in size and growth. These results are important for designing and understanding future studies of neuroprotection with high-dose rEPO.

Chronic Intra-Uterine Ureaplasma parvum Infection Induces Injury of the Enteric Nervous System in Ovine Fetuses.

Background: Chorioamnionitis, inflammation of the fetal membranes during pregnancy, is often caused by intra-amniotic (IA) infection with single or multiple microbes. Chorioamnionitis can be either acute or chronic and is associated with adverse postnatal outcomes of the intestine, including necrotizing enterocolitis (NEC). Neonates with NEC have structural and functional damage to the intestinal mucosa and the enteric nervous system (ENS), with loss of enteric neurons and glial cells. Yet, the impact of acute, chronic, or repetitive antenatal inflammatory stimuli on the development of the intestinal mucosa and ENS has not been studied. The aim of this study was therefore to investigate the effect of acute, chronic, and repetitive microbial exposure on the intestinal mucosa, submucosa and ENS in premature lambs. Materials and Methods: A sheep model of pregnancy was used in which the ileal mucosa, submucosa, and ENS were assessed following IA exposure to lipopolysaccharide (LPS) for 2 or 7 days (acute), Ureaplasma parvum (UP) for 42 days (chronic), or repetitive microbial exposure (42 days UP with 2 or 7 days LPS). Results: IA LPS exposure for 7 days or IA UP exposure for 42 days caused intestinal injury and inflammation in the mucosal and submucosal layers of the gut. Repetitive microbial exposure did not further aggravate injury of the terminal ileum. Chronic IA UP exposure caused significant structural ENS alterations characterized by loss of PGP9.5 and S100ß immunoreactivity, whereas these changes were not found after re-exposure of chronic UP-exposed fetuses to LPS for 2 or 7 days. Conclusion: The in utero loss of PGP9.5 and S100ß immunoreactivity following chronic UP exposure corresponds with intestinal changes in neonates with NEC and may therefore form a novel mechanistic explanation for the association of chorioamnionitis and NEC.

Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair.

Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock.

Latent Phase Identification of High-Frequency Micro-Scale Gamma Spike Transients in the Hypoxic Ischemic EEG of Preterm Fetal Sheep Using Spectral Analysis and Fuzzy Classifiers.

Premature babies are at high risk of serious neurodevelopmental disabilities, which in many cases are related to perinatal hypoxic-ischemic encephalopathy (HIE). Studies of neuroprotection in animal models consistently suggest that treatment must be started as early as possible in the first 6 h after hypoxia-ischemia (HI), the so-called latent phase before secondary deterioration, to improve outcomes. We have shown in preterm sheep that EEG biomarkers of injury, in the form of high-frequency micro-scale spike transients, develop and evolve in this critical latent phase after severe asphyxia. Real-time automatic identification of such events is important for the early and accurate detection of HI injury, so that the right treatment can be implemented at the right time. We have previously reported successful strategies for accurate identification of EEG patterns after HI. In this study, we report an alternative high-performance approach based on the fusion of spectral Fourier analysis and Type-I fuzzy classifiers (FFT-Type-I-FLC). We assessed its performance in over 2520 min of latent phase EEG recordings from seven asphyxiated in utero preterm fetal sheep exposed to a range of different occlusion periods. The FFT-Type-I-FLC classifier demonstrated 98.9 ± 1.0% accuracy for identification of high-frequency spike transients in the gamma frequency band (namely 80-120 Hz) post-HI. The spectral-based approach (FFT-Type-I-FLC classifier) has similar accuracy to our previous reverse biorthogonal wavelets rbio2.8 basis function and type-1 fuzzy classifier (rbio-WT-Type-1-FLC), providing competitive performance (within the margin of error: 0.89%), but it is computationally simpler and would be readily adapted to identify other potentially relevant EEG waveforms.

Plasma vasopressin levels are closely associated with fetal hypotension and neuronal injury after hypoxia-ischemia in near-term fetal sheep.

BACKGROUND: Sensitive biomarkers are needed to rapidly identify high-risk infants after hypoxia-ischemia for neuroprotective treatment. Hypotension is a key determinant of hypoxic-ischemic neural injury, and a potent stimulus of humoral pressors including angiotensin-II and arginine vasopressin. We therefore aimed to quantify the relationship between vasopressin and angiotensin-II levels in the latent phase after hypoxia-ischemia induced by umbilical cord occlusion (UCO) with both the severity of preceding hypotension and subsequent neuronal injury. METHODS: Chronically instrumented near-term fetal sheep underwent sham-UCO or UCO for either 15 min or until mean arterial pressure was <8 mmHg. Neuronal injury was assessed after 72 h recovery. RESULTS: Umbilical cord occlusion was associated with severe hypotension that recovered after UCO; two fetuses developed profound secondary hypotension within 6 h and died. Vasopressin levels but not angiotensin-II were significantly elevated 1-3 h after UCO and were closely associated with the severity of hypotension during UCO and the subsequent severity of neuronal loss in the parasagittal and lateral cortex, caudate nucleus and putamen. The Youden cut-point for vasopressin at 1 h was 180.0 pmol/L, with sensitivity 100% and specificity 92.3% for severe neuronal injury or death. CONCLUSION: Vasopressin levels shortly after moderate-severe hypoxia-ischemia may be a useful early biomarker to guide the timely implementation of neuroprotective treatment. IMPACT: It can be difficuIt to rapidly identify infants who might benefit from therapeutic hypothermia. We investigated whether increases in plasma pressor hormones early after hypoxia-ischemia were biomarkers for neonatal hypoxic-ischemic encephalopathy using near-term fetal sheep. Arginine vasopressin levels were elevated at 1-3 h after hypoxia-ischemia and were predictive of the severity of preceding hypotension and subsequent risk of severe neuronal injury or death after hypoxia-ischemia. Arginine vasopressin may help identify neonates at high risk of hypoxic-ischemic encephalopathy early within the therapeutic window for hypothermia.