Paul Maclean

Background: Methane emissions from enteric fermentation in ruminant livestock make up 27% of anthropogenic methane emissions. Results: Screening > 1,700 lactic acid bacteria identified Lacticaseibacillus rhamnosus FNZ118 (Kowbucha™ FNZ118) and L. rhamnosus FNZ142, (Kowbucha™ FNZ142) as capable of inhibiting rumen methanogens and methane production in vitro. FNZ118 or FNZ142 fed daily to Friesian heifer calves from birth to 14 weeks substantially lowered methane production through the first year of life compared to control animals. These strains also decreased feed intake and reduced ruminal metabolite concentrations without affecting animal live weight, suggesting an improvement in feed conversion efficiency. The observed effects did not cause major changes in the structure of the rumen microbiome. Conclusions: These findings demonstrate that early life provision of specific L. rhamnosus strains lower CH ₄ production and have potential for the mitigation of enteric greenhouse gas emissions from growing dairy cattle.

Epichloë fungal endophytes are an important symbiont of some temperate forage grasses, providing the plant host with protection from biotic and abiotic stresses. Recent research has focussed on applying the potential benefits of the asexual form of this endophyte to cultivated cereal crops. Cereals are not known to naturally host Epichloë endophytes but can be artificially inoculated with selected fungal strains isolated from Elymus and Hordeum species. A critical aspect of a successful association is the ability of these obligate mutualistic endophytes to transmit through the seed and colonise the resulting offspring. Within agricultural and cropping systems, seed can be stored for varying lengths of time before being sown. The ability of these fungal endophytes to survive in stored seed and colonise the next generation of plants is an important consideration. Optimal storage conditions required for Epichloë survival in seed of perennial ryegrass (Lolium perenne L.) and tall fescue (Festuca arundinacea Schreb.) are well documented, but little is known about endophyte survival within stored cereal grain, nor the conditions that are required for maximising its survival. Rye (Secale cereale L.) grain colonised with endophyte strain AR3002 and perennial ryegrass seed colonised with AR1 endophyte were stored under different temperature and humidity conditions, with the survival of both host and endophyte evaluated over a 2-year period. Under controlled conditions of 0°C and a relative humidity of 24%, the endophyte and host maintained stable levels of viability for both species for the duration of the trial. In environments where both the temperature and relative humidity were not controlled and the seed was exposed to ambient conditions, the endophyte generally declined rapidly within 12-18 months, although in most cases the seed itself remained viable, for both rye and perennial ryegrass seed. The endophyte associated with rye responded in a similar way to endophyte associated with perennial ryegrass in these storage environments. This understanding provides guidance on seed storage conditions required to ensure sustained endophyte viability but also identifies timeframes in which endophytic grain will need to be resown if held at ambient conditions. Further work will be required to determine if similar limitations are applicable to Epichloë endophytes in wheat.

Epichloë festucae var. lolii and Epichloë sp. LpTG-3 are filamentous fungal endophytes of perennial ryegrass (Lolium perenne) that have a substantial impact on New Zealand’s agricultural economy by conferring biotic advantages to the host grass. Epichloë endophyte strain AR37 provides ryegrass with improved agronomic performance, insect protection and plant persistence. Indole diterpenes are a class of well characterised secondary metabolites produced by Epichloë endophytes, with the AR37 strain expressing epoxyjanthitrems, a class of decorated indole diterpenes. These have been associated with the observed effects of AR37 on livestock and insect pests. Here we used gene inactivation by CRISPR-Cas9 to deconvolute the genetic basis for epoxyjanthitrem biosynthesis and manipulate this secondary metabolite pathway to reduce or remove endophyte-induced mammalian toxicity whilst retaining activity against some important agricultural insect pests. We also show that gene editing of Epichloë can be achieved without off-target events or introduction of foreign DNA (footprint-less) and that these gene edits can be classified as site-directed nuclease 1 (SDN-1), which are not regulated as genetically modified organisms in selected jurisdictions, including Australia. These SDN-1 technology edits have been successfully grown out of containment in the field in Australia for seed multiplication, followed by agronomy trials, which are now being evaluated. This has the potential to provide a step change in the future use of animal safe Epichloë strains in New Zealand pastures which will significantly reduce chemical inputs and increase animal welfare.

Introduction: In order to understand the emergence, persistence, and transmission of Campylobacter jejuni in livestock, this longitudinal study characterized the C. jejuni population in young calves and assessed the impact of exposure to wild birds during the pre-weaning period. Methods and results: Faecal samples were collected on eight occasions from 48 calves housed in three pens between birth and 10 weeks of age, two of the pens being covered with orchard netting. From the 250 C. jejuni isolates obtained from the positive faeces, seven distinct sequence types (7-gene legacy MLST) were identified, with high genetic similarity of circulating strains. On each pen, sequential dominance of ST was observed, with ST-508 and ST-50 prevalent in calves under 4 weeks old, and ST-520 or ST-422 prevalent in the 8- to 10-week-old calves. Exposure of calves to wild birds did not influence genotypes distribution, α-diversity, and community dissimilarity. However, a difference in C. jejuni populations between the two groups of calves that were not exposed to birds suggests that calving rank may shape the type and succession of ST detected over time. Conclusion: This study provided no evidence for exposure to wild birds being a driver for C. jejuni population changes in healthy pre-weaned calves.

Reducing greenhouse gas (GHG) emissions from ruminants while maintaining economic viability is critical for the sustainability of the livestock industry. We used a modelling approach to examine the effects of a reproductive technology on farm GHG emissions and economic farm surplus (EFS). The use of an androstenedione vaccine enhances ewe productivity (20% increase in lambing rate) and can potentially have a small direct impact on ewe methane production (5% reduction). Eight Beef + Lamb NZ Economic Service Monitor farms were modelled in Farmax ^TM . As sole effects, a 5% reduction in methane emissions by breeding ewes resulted in a 2.0% reduction in GHG emissions, whereas a 20% increase in lambing rate increased GHG emissions by 5%. This declined to 3.9% when both effects were considered. Increasing lambing by 20% without and with ewe numbers adjusted to maintain similar intake to Baseline, increased EFS by 74% and 40%. However, without changing ewe numbers, feed supply did not meet demand. Both effects combined with ewe numbers adjusted to maintain Baseline EFS resulted in a 7% reduction in GHG emissions. The study highlights the importance of investing in technologies that lead to increases in productivity, while the search for mechanisms to reduce emissions continues.

The production of structured triacylglycerols (STAGs) enriched in sn-2 specific fatty acids were examined for the microalgae Chlamydomonas reinhardtii, Scenedesmus obliquus, Nannochloropsis oceanica, and Nannochloropsis oculata. The objective was to explore the effects of varied cultivation conditions—including nutrient (nitrogen and phosphorus) starvation, and light saturation—on triacylglycerol (TAG) accumulation and the resultant fatty acid profiles. The total TAG content and specific fatty acid distribution, particularly focusing on the sn-2 position, were determined using an accelerated solvent extraction method and quantification by high-performance liquid chromatography and gas chromatography. Nutrient limitation significantly enhanced total lipid (up to two to threefold increase) and TAG accumulation across all species, with nitrogen starvation leading to the highest increases (C. reinhardtii = 172.99 ± 18.55 mg/g; S. obliquus = 221.65 ± 0.91 mg/g; N. oculata = 128.066 ± 8.37 mg/g; and N. oceanica = 211.96 ± 18.83 mg/g). Among the three types of sn-2 fatty acids (C16:0, C18:1, and C18:2) elevated levels of sn-2 palmitate (C16:0) = 64%–85% were recorded for both the Nannochloropsis strains under nitrogen- and phosphorus-limiting conditions, whereas under nitrogen-limiting conditions, around 56.8% and only 18% of sn-2 palmitate were recorded for C. reinhardtii and S. obliquus, respectively. Considering the total TAG content and higher cellular levels of sn-2 palmitate, both Nannochloropsis and C. reinhardtii strains could be promising candidates for natural STAG production. These findings provide valuable insight into leveraging microalgae for high-value lipid ingredients, paving the way for sustainable applications in clinical nutrition and infant formula development.

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.