Current livestock breeding is slow to respond to rapidly mounting environmental pressures that threaten sustainable animal protein production. New approaches can accelerate genetic improvement by multiplying valuable embryonic, rather than adult genotypes. Chimeras, derived from complementing a sterile host with a fertile donor embryo, provide a pathway to multiply and exclusively transmit elite male germlines. We established genetically sterile hosts and optimized embryo complementation conditions to achieve absolute germline transmission in sheep. The spermatogonia-specific gene NANOS2 was disrupted in male (NANOS2+/−, NANOS2−/−) and female (NANOS2−/−) ovine fetal fibroblasts via gRNA–Cas9-mediated homology-directed repair. Targeted cell strains and wild-type controls were used to produce cloned offspring for breeding and phenotyping. Male homozygous knockout clones lacked detectable germ cells, while the somatic compartment of the testis remained intact. By contrast, male monoallelic and female biallelic targeting of NANOS2 did not affect germline development, resulting in fertile animals capable of producing fertile offspring with normal reproductive performance. The germ cell niche in NANOS2−/− hosts was most efficiently complemented by aggregating compacted morulae, rather than earlier cleavage stages, resulting in 97% blastocyst chimerization. Embryo-complemented cloned lambs from two different donor cell lines showed variable chimerism across tissues from each germ layer, including various degrees of germline colonization. A subset of germline chimeras contained normal numbers of prospermatogonia, indicating that the germline was fully restored for absolute transmission of the donor cell genotype.

Background: Long COVID (LC) causes persistent symptoms, including fatigue, Musculo-skeletal (MSK) pain, and a lower quality of life. It's hypothesised that chronic low-grade inflammation in LC could impact bone, joints, and muscle microcirculation, but evidence is limited. Aim: To assess health-related quality of life (HRQoL) and circulating inflam-mation, bone turnover markers (BTM), and vitamin D in LC individuals to explore their potential association with MSK function. Methods: A longitudinal prospective cohort study involving 45 adults with LC and 40 well-recovered (WR) post-COVID-19 controls was conducted over 12 months. Baseline and follow-up assessments included evaluations of HRQoL and pain rating questionnaires; blood analysis of inflammatory and bone turnover markers (BTM). Results: More females were in the LC group. LC reported significantly lower HRQoL compared to WR, with no change over 12 months. LC had higher Vitamin D levels at baseline compared to WR 15.9 vs 23.7 ng/ml (p=0.0021). Both groups experienced significant increases in Vitamin D after 12 months: WR from 21.4 to 28.5 ng/ml (p=0.0001) and LC from 32.6 to 35.8ng/ml (p=0.0023). LC also experienced more hand pain at baseline (p=0.003), which improved over time. No differences between groups in BTM or cytokines over time. Conclusion: Existing evidence suggests that this cohort study on LC adults and controls found a reduction in HRQoL and joint symptoms, indicating the need for ongoing monitoring. Future studies should explore MSK, muscle function via imaging, and ways to enhance musculoskeletal health and well-being.

Background/Objectives: Long COVID (LC) causes persistent symptoms, including fatigue, musculoskeletal (MSK) pain, and a lower quality of life. It is hypothesised that chronic low-grade inflammation in LC could impact bone, joints, and muscle microcirculation, but evidence is limited. Our aim is to assess health-related quality of life (HRQoL) and circulating inflammation, bone turnover markers (BTM), and vitamin D in LC individuals to explore their potential association with MSK function. Methods: Prospective longitudinal cohort; LC n = 45, well-recovered (WR) n = 40; 12 ± 2 months follow-up. Baseline and follow-up assessments included evaluations of HRQoL and pain-rating questionnaires, and blood analysis of inflammatory and bone turnover markers (BTM). Results: More females were in the LC group. LC reported significantly lower HRQoL compared to WR, with no change over 12 months. LC had higher vitamin D levels at baseline, median 29.46 ng/mL (23.75; 35.06) compared to WR 20.36 ng/mL (15.995; 27.65) (p = 0.0021). Both groups experienced significant increases in vitamin D after 12 months: WR median from 21.4 ng/mL (16.34; 27.89) to 29.58 ng/mL (25.33; 41.74), (p =< 0.001) and LC median from 32.695 ng/mL (23.665; 35.1) to 35.89 ng/mL (30.1; 41.2), (p = 0.0023). Pain rating showed LC also experienced more hand pain at baseline median 1 (0; 5), (p = 0.003). There were no differences between groups in BTM or cytokines over time. Conclusions: This feasibility cohort showed that LC is associated with a reduction in HRQoL and joint symptoms; however, no significant changes were observed in the inflammatory markers, indicating the need for ongoing monitoring. Future studies should explore MSK, muscle function via imaging, and ways to enhance musculoskeletal health and well-being.

Livestock have long been regarded as a potential source of donor organs to alleviate the global organ shortage for transplantation. Sheep have a similar anatomy to humans, providing the standard model for demonstrating biocompatibility and performance of biological heart valves to obtain regulatory approval for their use in transplantation. Like most mammals, sheep cells contain two well‐characterized carbohydrate epitopes, galactose‐α1,3‐galactose (α‐Gal) and N ‐glycolylneuraminic acid (Neu5Gc), which are absent in humans. Formation of these xenoantigens is catalyzed by two enzymes, namely α1,3 galactosyltransferase (GGTA1) and CMP‐Neu5Gc hydroxylase (CMAH), respectively. Towards generating new sheep models, we used Cas9‐mediated genome editing in both male and female ovine fetal fibroblasts to knockout (KO) both alleles of CMAH and GGTA1 . Selected double KO (DKO) fibroblast strains were used for somatic cell transfer cloning to produce blastocysts that were transferred into gestational surrogate ewes. Following the transfer of 128 male and 40 female cloned blastocysts, six male and eight female lambs were born; however, none of the males survived. Molecular analyses of cells from the five surviving ewes confirmed their compound heterozygous state, resulting in a functional DKO phenotype and subsequent immune rejection of embryonic tissues during natural breeding with wild‐type rams. This new DKO sheep model better mimics the human immune status, offering greater physiological relevance for preclinical testing of biological heart valves, alleviation of red meat allergy syndrome due to the presence of dietary xenoantigens, and a widely culturally acceptable alternative source of certain donor organs.

This protocol summarises zona-free sheep cloning by somatic cell transfer (SCT), adapted from a similar SCT method in cattle (https://doi.org/10.1089/153623003772032763). It describes the workflow that is required for reprogramming somatic cells into cloned blastocysts of high morphological quality, including two optional steps of embryo aggregation and vitrification. The resulting embryos can then be transferred into surrogate ewes for producing live cloned animals. Compared to conventional zona-intact procedures, this zona-free embryo reconstruction system increases throughput of cloned embryo production and ease of operation.