Fetal mortality significantly impacts reproductive efficiency in sheep, with approximately 30% of fetuses failing to survive until lambing. In multifetal gestation, partial litter loss (PLL) occurs in 28% to 37% of cases. The biochemical composition of allantoic fluid (ALF), which plays a key role in metabolic regulation and fetal waste clearance, has been shown in other species to reflect fetal development and maturity. However, ALF metabolomics in sheep, especially in relation to PLL, remains unexplored. This study investigated late-gestation fetal mortality in multifetal sheep pregnancies by analyzing maternal metabolic status, placental gene expression, and ALF composition to identify potential metabolic compounds associated with PLL. The study was conducted on 25 ewes, of which 14 experienced PLL, and 11 had vital litters (VL). Plasma analysis revealed that VL ewes have higher β-hydroxybutyrate concentrations than PLL ewes, whereas plasma insulin and cortisol levels are higher in PLL ewes. Analysis of gene expression in the placental cotyledon demonstrated increased androgen receptor expression in PLL pregnancies and trends toward elevated solute carrier family 2 member 1, and reduced nuclear receptor subfamily 3 group C member 1 expression. Insulin-like growth factor 2 expression tended to be higher in VL placentas. Allantocentesis was performed on 25 ewes with multifetal pregnancies (11 VL and 14 PLL). ALF samples were collected from 74 fetuses at 140.4 ± 2.0 days in pregnancy, and 41 samples were selected for metabolomic profiling. Using genomic analysis, 25 ALF samples were confidently matched to individual lambs. Untargeted lipidomics of ALF from 14 lambs in PLL (7 born alive vs. 7 stillborn) revealed 24 differentially abundant lipid compounds. Stillborn lambs exhibited lower phosphatidylglycerol (PG) 15:0_20:1, but higher levels of sphingomyelin (SM), lysophosphatidylcholine (LPC), ether-linked phosphatidylethanolamines (PEs), lysophosphatidylethanolamines (LPEs), and monogalactosyldiacylglycerols (MGDGs). Collectively, PLL in multifetal ewe pregnancies is linked to maternal metabolic imbalance, elevated cortisol levels, dysregulated placental gene expression, and altered ALF lipid profiles in stillborn fetuses. Elevated SM and LPC, and lower PG, suggest immature lungs. These molecular and metabolic signatures may provide new insights into the mechanisms underlying PLL in late-gestation multifetal pregnancies.

SGLT2 inhibitors (SGLTi) confer significant cardiometabolic benefits in diabetes, but are associated with increased risk of diabetic ketoacidosis (DKA). We hypothesized that supporting anaplerosis with glycerol could mitigate SGLTi-induced ketosis, enhance insulin-mediated resolution of severe diabetic ketosis, and lower the risk of DKA when used as a dietary supplement. Insulin-dependent diabetes was induced in yearling sheep using alloxan. The animals subsequently developed severe hyperglycemia (glucose > 500 mg/dL) and hyperketonemia (β-hydroxybutyrate [BHB] > 5 mM), requiring daily insulin therapy for metabolic control and survival. Ketosis was induced by partial insulin withdrawal and with the administration of 10 mg of the SGLT2 inhibitor empagliflozin. Animals were randomly assigned in a cross-over trial to evaluate the effects of intravenous 5

Ketosis and its severe complication in small ruminants, pregnancy toxemia, are major metabolic disorders characterized by hypoglycemia and high blood concentrations of β-hydroxybutyrate (BHBA) resulting from negative energy balance (NEB). Although conventional treatments rely on glucogenic precursors like glycerol and propylene glycol, farmers often turn to lower-cost alternatives, including sugary soft drinks containing high-fructose corn syrup (HFCS) and carbonated water (cWater). In this study, we evaluated the relative contribution of the primary components of Coca-Cola (Coke), used here as a representative and readily accessible energy source, in mitigating NEB in sheep. Twenty-eight Afec-Assaf ewe lambs (N = 28), fasted for 48 h to induce NEB, were randomly assigned to four treatment groups (n = 7): 1 L of either Water, cWater, carbonated HFCS solution (cHFCS), or Coke. Blood samples were collected at 0 and 1, 2, and 3 h post-treatment to measure glucose and BHBA concentrations. Glucose area under the curve (AUC) values were highest in cHFCS (2339.5 ± 628.6 mg/dL × min) and Coke (1778.5 ± 437.6 mg/dL × min), intermediate in cWater (950.8 ± 391.3 mg/dL × min), and lowest in Water (570.0 ± 184.5 mg/dL × min). ANOVA indicated significant treatment effects (P <0.037), with post-hoc analysis showing that cHFCS yielded higher glucose AUC than Water (P <0.05). BHBA AUC also differed significantly among treatments (P <0.018), with greater effects for cHFCS (-4.72 ± 4.55 mM × min) and Coke (-5.59 ± 2.96 mM × min) compared with Water (-20.98 ± 5.1 mM × min) and cWater (-21.02 ± 5.12 mM × min). Pairwise comparisons showed no significant differences between cHFCS and Coke, but indicated a trend (P = 0.08) toward a greater response to cHFCS compared to Water and cWater. Overall, the results identify HFCS as the primary component of soft drinks responsible for improving NEB, suggesting that HFCS could represent a practical and economical energy supplement for on-farm ketosis management.

Pregnancy toxemia (PT) is the most frequent metabolic disorder for ewes in late pregnancy. Although propylene glycol (PG) and glycerol (GLY) are common glycogenic supplements for treating PT in ewes, the relative benefit of these supplements is not clear. In a previous study, it was demonstrated that PG was mainly effective in reducing the β-hydroxybutyrate acid (BHB) concentration, whereas GLY was more effective in increasing the plasma glucose concentration. Therefore, the objectives of the present study were to determine the effects of drenching various doses and combinations of PG and GLY to late-pregnant ewes in order to obtain a simultaneous increase in glucose and a decrease in BHB. Sixty-six multiparous Afec-Assaf ewes bearing at least two fetuses were used in 3 consecutive experiments. In all experiments, blood samples were collected twice before drenching (at 0630 and 0645h), then the ewes were drenched with each supplement at 0700h, and blood samples were taken every hour for 12 to 14h post-drenching. In experiment 1, ewes were drenched either with 1) Control (CTL1, n = 6) – 55mL water, 2) PG100 (n = 6) – 106mL of PG, 3) GLY100 (n = 6) –108mL of Koforin (containing 80% glycerol, 15% water, and 5% ash), 4) PG50 (n = 6) – 53mL of PG, and 5) GLY50 (n = 6) – 54mL of Koforin. The plasma glucose concentration was higher after the GLY50 (P = 0.03) and GLY100 (P = 0.01) treatments than in CTL1. The plasma BHB concentration was lower after the PG50 (P = 0.001) and GLY50 (P = 0.02) treatments and tended to be lower in the PG100-treated ewes (P = 0.10) than in the CTL1 ewes. In experiment 2, ewes were drenched with 1) Control (CTL2, n = 6) –55mL water, 2) MIX50 (n = 6) –26.5mL PG + 27mL Koforin, and 3) MIX100 (n = 6) –53mL PG + 54mL Koforin. No differences were observed among treatments for the main blood metabolites. In experiment 3, ewes were drenched with 1) Control (CTL3, n = 6) – 110mL water, 2) MIX200(1:1) (n = 6) –106mL PG +108mL Koforin, and 3) MIX200(3:7) (n = 6) – 63.4mL PG +150.6mL Koforin. The plasma glucose concentrations were 17.8% and 20.9%, respectively, higher in the MIX200(1:1) and MIX200(3:7) ewes than in the CTL3 ewes (P < 0.01). The plasma BHB concentrations in the MIX200(1:1) and MIX200(3:7) ewes were 42% and 47%, respectively, lower than in the CTL3 ewes (P < 0.03). The NEFA concentration in the MIX200(1:1) and MIX200(3:7) ewes was 60% and 57%, respectively, lower than in the CTL3 ewes (P = 0.008). In conclusion, MIX200(1:1) and MIX200(3:7) were the most effective approaches to increasing glucose and reducing the BHB and NEFA concentrations concomitantly in the plasma of late-pregnant ewes.

Gene augmentation therapy is a promising treatment for incurable, blinding inherited retinal diseases, and intravitreal delivery is being studied as a safe alternative to subretinal injections. Adeno-Associated Viruses (AAV) are commonly-used vectors for ocular gene augmentation therapy. Naturally occurring pre-operative exposure and infection with AAV could result in presence of neutralizing antibodies (NAB’s) in patients’ serum, and may affect the safety and efficacy of treatment. Our aim was to characterize the humoral response against AAV pre- and post-intravitreal delivery of AAV2.7m8 vectors in a naturally-occurring sheep model of CNGA3 achromatopsia. Serial serum neutralization assays were performed to screen sheep for pre-exiting anti-AAV2 NAB’s, and to assess the effect of intravitreal AAV2.7m8 injection on post-operative NAB titers and intraocular inflammation in sheep. The effect of viral dose and transgene type were also assessed. Serological screening revealed pre-operative seropositivity in 21.4% of animals, with age being a risk factor for the presence of anti-AAV2 NAB’s. NAB titers increased following intravitreal AAV administration in the majority of sheep. There was no significant difference in the degree of post-operative serum neutralization between pre-operatively seronegative sheep and those with pre-existing antibodies. However, only sheep with pre-existing antibodies presented with signs of post-operative inflammation. We conclude that pre-existing anti-AAV2 NAB’s do not affect the level of post-operative NAB titers; however, they increase the risk of post-operative ocular inflammation. Our results could have implications for the management of AAV-mediated ocular gene therapies, a technology being increasingly studied and used in patients.

In sheep, ~30% of fetuses do not survive till parturition, and 17.7% of the multifetal pregnancies experience partial litter loss (PLL). In humans, multifetal pregnancies are associated with a higher risk of perinatal mortality. Therefore, the objectives were to examine the association between partial litter loss, fetal sex, dam’s metabolic and physiological state, and pregnancy outcome in multifetal pregnant ewes. The study includes two parts. The first was a retrospective study, in which we analyzed data of 675 lambings and examined the PLL incidence according to male ratio (MR) for all litter sizes (range 2–6). Lambings were categorized as having a low male ratio (LMR; <50% males) or a high male ratio (LMR; >50% males). In the second part, we monitored 24 ewes from 80 to 138 days in pregnancy every 10 days, and then daily until lambing, by ultrasound scanning for maternal heart rate (HR), and Doppler ultrasound for litter vitality. Blood samples were taken from dams on the days of scanning. Male ratio strongly affected PLL, where the general survival rate (for all lambings) was reduced from 90% in LMR lambings to 85% in HMR lambings. The odds ratio for PLL in HMR vs. LMR litters was 1.82. Birth body weight and the survival rate of female was higher in LMR than HMR lambings, with no differences for male lambs in both parameters. In the second part, dams’ HR during the last trimester was 9.4% higher in LMR than in HMR pregnancies, with no differences in fetuses’ HR. The plasma glucose and insulin concentrations were not significantly different between groups, but plasma β-hydroxybutyrate and nonesterified fatty acid concentrations were, respectively, 31% and 20% lower in HMR vs. LMR ewes. In conclusion, male fetuses negatively affect pregnancy outcomes and influence dams’ metabolic and physiological state in sheep.

As a precursor for a universal metabolic coenzyme, vitamin B1, also known as thiamine, is a vital nutrient in all living organisms. We previously found that high-dose thiamine therapy prevents overnutrition-induced hepatic steatosis in sheep by enhancing oxidative catabolism. Based on this capacity, we hypothesized that thiamine might also reduce whole-body fat and weight. To test it, we investigated the effects of high-dose thiamine treatment in sheep under overnutrition and calorie-restricted undernutrition to respectively induce positive energy balance (PEB) and negative energy balance (NEB). Eighteen mature ewes were randomly assigned to three treatment groups (n = 6 each). The control group (CG) was administered daily with subcutaneous saline, whereas the T5 and T10 groups were administered daily with equivoque of saline containing 5 mg/kg and 10 mg/kg of thiamine, respectively. Bodyweight and blood biochemistry were measured twice a week for a period of 22 days under PEB and for a consecutive 30 days under NEB. Surprisingly, despite the strong effect of thiamine on liver fat, no effect on body weight or blood glucose was detectable. Thiamine did, however, increase plasma concentration of non-esterified fatty acids (NEFA) during NEB (575.5 ± 26.7, 657.6 ± 29.9 and 704.9 ± 26.1 µEqL−1 for CG, T5, and T10, respectively: p < 0.05), thereby favoring utilization of fatty acids versus carbohydrates as a source of energy. Thiamine increased serum creatinine concentrations (p < 0.05), which paralleled a trending increase in urea (p = 0.09). This may indicate an increase in muscle metabolism by thiamine. Reduction of fat content by thiamine appears more specific to the liver than to adipose tissue. Additional studies are needed to evaluate the potential implications of high-dose vitamin B1 therapy in muscle metabolism.

Sheep carrying a mutated CNGA3 gene exhibit diminished cone function and provide a naturally occurring large animal model of achromatopsia. Subretinal injection of a vector carrying the CNGA3 transgene resulted in long-term recovery of cone function and photopic vision in these sheep. Research is underway to develop efficacious vectors that would enable safer transgene delivery, while avoiding potential drawbacks of subretinal injections. The current study evaluated two modified vectors, adeno-associated virus 2-7m8 (AAV2-7m8) and AAV9-7m8. Intravitreal injection of AAV2-7m8 carrying enhanced green fluorescent protein under a cone-specific promoter resulted in moderate photoreceptor transduction in wild-type sheep, whereas peripheral subretinal delivery of AAV9-7m8 resulted in the radial spread of the vector beyond the point of deposition. Intravitreal injection of AAV2-7m8 carrying human CNGA3 in mutant sheep resulted in mild photoreceptor transduction, but did not lead to the clinical rescue of photopic vision, while day-blind sheep treated with a subretinal injection exhibited functional recovery of photopic vision. Transgene messenger RNA levels in retinas of intravitreally treated eyes amounted to 4–23% of the endogenous CNGA3 levels, indicating that expression levels >23% are needed to achieve clinical rescue. Overall, our results indicate intravitreal injections of AAV2.7m8 transduce ovine photoreceptors, but not with sufficient efficacy to achieve clinical rescue in CNGA3 mutant sheep.

Fatty liver is an abnormal metabolic condition of excess intrahepatic fat. This condition, referred to as hepatic steatosis, is tightly associated with chronic liver disease and systemic metabolic morbidity. The most prevalent form in humans, i.e. non-alcoholic fatty liver, generally develops due to overnutrition and sedentary lifestyle, and has as yet no approved drug therapy. Previously, we have developed a relevant large-animal model in which overnourished sheep raised on a high-calorie carbohydrate-rich diet develop hyperglycemia, hyperinsulinemia, insulin resistance, and hepatic steatosis. Here, we tested the hypothesis that treatment with thiamine (vitamin B1) can counter the development of hepatic steatosis driven by overnutrition. Remarkably, the thiamine-treated animals presented with completely normal levels of intrahepatic fat, despite consuming the same amount of liver-fattening diet. Thiamine treatment also decreased hyperglycemia and increased the glycogen content of the liver, but it did not improve insulin sensitivity, suggesting that steatosis can be addressed independently of targeting insulin resistance. Thiamine increased the catalytic capacity for hepatic oxidation of carbohydrates and fatty acids. However, at gene-expression levels, more-pronounced effects were observed on lipid-droplet formation and lipidation of very-low-density lipoprotein, suggesting that thiamine affects lipid metabolism not only through its known classic coenzyme roles. This discovery of the potent anti-steatotic effect of thiamine may prove clinically useful in managing fatty liver-related disorders. This article has an associated First Person interview with the joint first authors of the paper.

Fatty liver represents a significant metabolic pathology of excess intrahepatic fat in domestic animals and humans. Quantification of hepatic-fat content is therefore essential for diagnosis and investigation of liver and metabolic disease. However, the reproducibility of hepatic steatosis analysis is often low due to subjective and technical factors. We hypothesized that improvement in tissue-lipids extraction efficiency would contribute to the accuracy and precision of liver-fat determination. To test it, we investigated the effect of standardized tissue sonication on liver-fat quantification by the Folch method in sheep. Liver samples from grownup lambs of lean (n = 16) and fatty (n = 15) livers, and from pregnant ewes (n = 6) who died from pregnancy toxemia (PT), were used for hepatic-fat content determination with or without tissue sonication. In the grown lambs, an average hepatic-fat content of 6.6% was determined in sonicated compared to 5.1% in non-sonicated specimens (P = 0.0002). Similarly, in ewes with PT, an average of 12.5% was determined with sonication compared to 10.8% without it (P = 0.0006), and the reproducibility was higher with sonication (CV of 3.1 vs. 6.1%, respectively). Thus, tissue sonication improved the efficiency of liver-lipids extraction and was significant to the accuracy and precision of hepatic-fat determination. Enzymatic quantification of triglycerides was moderately correlated with the results obtained gravimetrically (r = 0.632, P < 0.005). The reported data provide reliable reference values for pregnancy toxemic sheep. The significant improvement in liver-fat quantification observed with the reported revised protocol is likely applicable to most mammals and humans.

Gene augmentation therapy based on subretinal delivery of adeno-associated viral (AAV) vectors is proving to be highly efficient in treating several inherited retinal degenerations. However, due to potential complications and drawbacks posed by subretinal injections, there is a great impetus to find alternative methods of delivering the desired genetic inserts to the retina. One such method is an intravitreal delivery of the vector. Our aim was to evaluate the efficacy of two capsid-modified vectors that are less susceptible to cellular degradation, AAV8 (doubleY-F) and AAV2 (quadY-F+T-V), as well as a third, chimeric vector AAV[max], to transduce photoreceptor cells following intravitreal injection in sheep. We further tested whether saturation of inner limiting membrane (ILM) viral binding sites using a nonmodified vector, before the intravitreal injection, would enhance the efficacy of photoreceptor transduction. Only AAV[max] resulted in moderate photoreceptor transduction following intravitreal injection. Intravitreal injection of the two other vectors did not result in photoreceptor transduction nor did the saturation of the ILM before the intravitreal injection. However, two of the vectors efficiently transduced photoreceptor cells following subretinal injection in positive control eyes. Previous trials with the same vectors in both murine and canine models resulted in robust and moderate transduction efficacy, respectively, of photoreceptors following intravitreal delivery, demonstrating the importance of utilizing as many animal models as possible when evaluating new strategies for retinal gene therapy. The successful photoreceptor transduction of AAV[max] injected intravitreally makes it a potential candidate for intravitreal delivery, but further trials are warranted to determine whether the transduction efficacy is sufficient for a clinical outcome.

Pregnancy toxemia is the most frequent metabolic disorder of ewes in late pregnancy. Although propylene glycol (PG) and glycerol (GLY) are common glucogenic supplements for treating pregnancy toxemia in ewes, the relative benefit of these 2 supplements is not entirely clear. Therefore, the objectives of the present study were to determine the changes during 24 h in key blood metabolites and insulin in response to PG or GLY drenching in prolific ewes. To this end, 36 multiparous late-pregnant Afec-Assaf ewes (∼132.4 d pregnant) bearing 2 to 4 fetuses, divided into 2 blocks (18 ewes in each block), with a blood β-hydroxybutyrate (BHB) concentration of 0.5 to 1.6 mmol/L were included. Ewes were divided into 3 groups (12 ewes each; 6 ewes in each experimental day), according to their BHB levels, expected litter size, body weight, and body condition score, and were drenched with the following: (1) control group (CTL), 55 mL of water; (2) PG, 106 mL of PG (100% PG, 448 calories); or (3) GLY, 108 mL of Koforin 80 (80% GL; 448 calories). Blood samples were taken before drenching and every hour after drenching for 24 h. Plasma concentration of glucose, BHB, nonesterified fatty acids, lactate, glycerol, and insulin were determined. Because there were no effects of treatments after 12 h in the first block, the data were analyzed for 12 h after drenching rather than 24 h. The plasma glucose concentration during the first 5 h after drenching was the highest in the GLY, BHB concentration was the lowest in the PG, and the nonesterified fatty acid levels were lower in the PG compared with the CTL ewes during the first 5 h after drenching. However, glucose concentration was higher in the PG ewes at 9, 11, and 12 h after drenching than in CTL or GLY ewes. The mean lactate concentration in plasma for 12 h was 2.5- and 1.9-fold higher in the PG compared with the CTL and GLY ewes, respectively, and except at 11 h after drenching, it was significantly higher at each time point. The insulin concentration was higher in the GLY than in both other groups at 2 to 5 h after drenching. These results suggest that during the first few hours after drenching the effect of PG was more effective in reducing the BHB concentration, whereas the GLY effect was more effective in enhancing glucose concentration. The increased concentration in lactate following PG treatment suggests that the PG contribution to gluconeogenesis is mediated through its metabolism to lactate. In contrast, the lack of an effect on lactate, and the faster increase in blood glucose in response to GLY suggest that GLY has a more advanced entry point to gluconeogenesis, which influences the immediate response in enhancing the glucose blood concentration.

Hepatic steatosis is strongly associated with chronic liver disease and systemic metabolic disorder. Adipose lipolysis is a recognized principal source of intrahepatic fat in various metabolic disorders, including non-alcoholic fatty liver disease. We hypothesized that, in the premorbid state, hepatic de novo lipogenesis (DNL) driven by excess carbohydrates abundance might play a more significant role. We employed a novel nutritional model in sheep of two distinct carbohydrates abundances. During 4 months of the dietary treatment, lambs were monitored for metabolic and terminal liver parameters. Lambs grown on the high-calorie (HC) diet were consistently more hyperglycemic and hyperinsulinemic than lambs grown on the lower-calorie (LC) diet (P<0.0001). As a result, the HC lambs developed systemic- (HOMA-IR of 7.3 vs. 3.1; P<0.0001), and adipose- (ADIPO-IR of 342.7 vs. 74.4; P<0.0001) insulin resistance, significant adiposity (P<0.0001), and higher plasma triglycerides (P<0.05). Circulating leukocytes in the HC lambs had higher mRNA expression levels of the proinflammatory markers CCL2 (P<0.01) and TNF-alpha (P<0.04), and IL1B trended higher (P<0.1). Remarkably, lambs on the HC diet developed substantial liver steatosis (mean fat content of 8.1 vs. 5.3% in the LC group; P<0.0001) with a higher histological steatosis score (2.1 vs. 0.4; P<0.0002). Hepatic steatosis was most-strongly associated with blood glucose and insulin levels but negatively correlated with circulating fatty acids-indicating a more significant contribution from hepatic DNL than from adipose lipolysis. Sheep may prove an attractive large-animal model of fatty liver and metabolic comorbidities resulting from excess carbohydrate-based energy early in life.

Achromatopsia is an inherited retinal disease characterized by loss of cone photoreceptor function. Day blind CNGA3 mutant Improved Awassi sheep provide a large animal model for achromatopsia. This study measured refractive error and axial length parameters of the eye in this model and evaluated chromatic pupillary light reflex (cPLR) testing as a potential screening test for loss of cone function. Twenty-one CNGA3 mutant, Improved Awassi, 12 control Afec-Assaf and 12 control breed-matched wild-type (WT) Awassi sheep were examined using streak retinoscopy and B-mode ocular ultrasonography. Four CNGA3 mutant and four Afec-Assaf control sheep underwent cPLR testing. Statistical tests showed that day-blind sheep are significantly more myopic than both Afec-Assaf and WT Awassi controls. Day-blind sheep had significantly longer vitreous axial length compared to WT Awassi (1.43 ± 0.13 and 1.23 ± 0.06 cm, respectively, p < 0.0002) and no response to bright red light compared to both controls. Lack of response to bright red light is consistent with cone dysfunction, demonstrating that cPLR can be used to diagnose day blindness in sheep. Day-blind sheep were found to exhibit myopia and increased vitreous chamber depth, providing a naturally occurring large animal model of myopia.

This research determined the effects of dietary supplementation with rumen-protected arginine (Pro-Arg) on metabolites and amino acids in maternal plasma and lamb survival rate at birth (LSRAB) in prolific Afec-Assaf ewes. The hypothesis was that Pro-Arg, the precursor for nitric oxide and polyamines, would increase placental development and vascularity, uteroplacental blood flow, and nutrient transport and reduce oxidative stress to increase LSRAB. Ewes were fed either their basal diet, basal diet with Pro-Arg, or basal diet with unprotected arginine (Unp-Arg; 18 g/head/d). The supplemental arginine was about 1% of the dry matter intake from day 40 or 60 of gestation until parturition. Ninety-two of 98 ewes produced live lambs. Ewes fed Pro-Arg had greater (P = 0.002) concentrations of arginine and other amino acids in plasma, whereas Unp-Arg did not affect concentrations of arginine, but decreased (P < 0.05) concentrations of some amino acids. There was no effect of treatments on gestation length (144 +/- 2 d), prolificacy (2.65 lambs born per ewe), LSRAB (0.80), body weight (88.8 +/- 10.8 kg), and body condition score (2.8 +/- 0.6) of ewes, or birth weight and crown-rump length of lambs. The GI (BW/CRL1.5) was affected by sex of lamb (P = 0.008), parity of ewe (P = 0.002), litter size (P = 0.0001), and lamb status (P = 0.003). Of 229 lambs born, 32 were dead and 16 died before 5 mo of age, leaving 181 lambs with records on weights at birth and 5 mo of age. Interestingly, lambs born to ewes fed the Unp-Arg and Pro-Arg weighed 3.6 kg less at postnatal day 150 than lambs from control ewes.

Negative energy balance (NEB) is a state of insufficient dietary-energy consumption, characterized by the breakdown of adipose fat to meet the physiological energy expenditure. Extensive NEB, as common in high-yielding transitioning ruminants, drives significant metabolic disturbance and pathologies such as pregnancy toxemia and ketosis. Strategies to minimize the severity of NEB include the use of energy-dense feed supplements, like glycerol and propylene glycol (PG), or IV glucose infusion during severe hypoglycemia. PG and glycerol have been studied mainly by oral or ruminal administration, which exposes them to substantial metabolism in the digestive system. To investigate their direct benefits to mitigating NEB, we intravenously infused them into sheep induced into NEB by feed restriction. Sixteen 5-month-old ewe lambs at NEB were IV-treated with 170 mL isotonic saline containing 15% glycerol or 15% PG. Both PG and glycerol effectively reduced hyperketonemia by 57% and 61%, and inhibited adipose lipolysis by 73.6% and 73.3%, respectively. Surprisingly, only glycerol was glucogenic (p < 0.0001) and insulinotropic (p < 0.0075), while PG was primarily utilized for production of lactate (p < 0.0001). Tissue-damage biomarkers indicated hemolytic activity for PG. This study revealed glycerol as a superior IV treatment for effective relief of NEB. Since it carries no risk of glucose overloading, glycerol IV infusion may also have clinical advantages over glucose for treatment of pregnancy toxemia and ketosis.

Our aim was to compare the electroretinographic (ERG) responses of two eyes obtained by consecutive unilateral recordings to those obtained by a simultaneous bilateral recording in sheep.

Achromatopsia causes severely reduced visual acuity, photoaversion, and inability to discern colors due to cone photoreceptor dysfunction. In 2010, we reported on day-blindness in sheep caused by a stop-codon mutation of the ovine CNGA3 gene and began gene augmentation therapy trials in this naturally occurring large animal model of CNGA3 achromatopsia. The purpose of this study was to evaluate long-term efficacy and safety results of treatment, findings that hold great relevance for clinical trials that started recently in CNGA3 achromatopsia patients. Nine day-blind sheep were available for long-term follow up. The right eye of each sheep was treated with a single subretinal injection of an Adeno-Associated Virus Type 5 (AAV5) vector carrying either a mouse (n = 4) or a human (n = 5) CNGA3 transgene under control of the 2.1-Kb red/green opsin promoter. The efficacy of treatment was assessed periodically with photopic maze tests and electroretinographic (ERG) recordings for as long as 74 months postoperatively. Safety was assessed by repeated ophthalmic examinations and scotopic ERG recordings. The retinas of three animals that died of unrelated causes >5 years post-treatment were studied histologically and immunohistochemically using anti-hCNGA3 and anti-red/green cone opsin antibodies. Passage time and number of collisions of treated sheep in the photopic maze test were significantly lower at all follow-up examinations as compared with pretreatment values (p = 0.0025 and p < 0.001, respectively). ERG Critical Flicker Fusion Frequency and flicker amplitudes at 30 and 40 Hz showed significant improvement following treatment (p < 0.0001) throughout the study. Ophthalmic examinations and rod ERG recordings showed no abnormalities in the treated eyes. Immunohistochemistry revealed the presence of CNGA3 protein in red/green opsin-positive cells (cones) of the treated eyes. Our results show significant, long-term improvement in cone function, demonstrating a robust rescue effect up to six years following a single treatment with a viral vector that provides episomal delivery of the transgene. This unique follow-up duration confirms the safe and stable nature of AAV5 gene therapy in the ovine achromatopsia model.