The development and assessment of novel non-invasive methods of measuring sleep in dairy cows : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Manawatū, New Zealand

Onet published article in Appendix C was removed for copyright reasons, but may be accessed via its source: Hunter, L.B., O’Connor, C., Haskell, M.J., Langford, F.M., Webster, J.R., & Stafford, K.J. 2021, September. Lying posture does not accurately indicate sleep stage in dairy cows. Applied Animal Behaviour Science, 242, 105427. https://doi.org/10.1016/j.applanim.2021.105427Sleep is important for animal health and welfare and there are many factors, for example, the environment, illness, or stress, that are likely to have an impact on cow sleep and consequently affect their welfare. Polysomnography (PSG) is considered the gold standard for precise identification of sleep stages. It consists of electrophysiological recordings of the brain activity, eye movements and muscle activity but is costly and difficult to use with cows on farm. Accordingly, the study of sleep in cows may be limited due to the impracticability of PSG. Alternative methods of assessing sleep have been developed for humans. Some such work has been conducted for cows, but this has yet to be validated with PSG. The main aim of this thesis was to investigate alternative methods to PSG to accurately detect sleep stages in dairy cows. Specifically, I aimed to develop a detailed 5-stage scoring system for assessing sleep stages from the cow PSG, to investigate the suitability of using lying postures and heart rate (HR) measures to assess sleep stages and to develop a model to accurately predict sleep stages using non-invasive measures in dairy cows compared with PSG. Two studies were conducted using 6 non-lactating dairy cows in an indoor housed environment in Scotland, and outdoors at pasture in New Zealand. PSG was recorded with each cow over a period of seven days. From these data a 5-stage sleep-scoring criteria with good reliability was developed which identified two stages of light sleep, two stages of deep sleep as well as awake and rumination stages. Video was recorded during sleep recordings and the cow’s behaviour was analysed and compared with the scored sleep stages from the PSG. Some sleep stages were found to occur mainly in specific lying postures; however, overall, postures were not useful indicators of sleep stages. Heart rate (HR) and heart rate variability (HRV) were measured using a Polar HR monitor ii and data logging device. Differences in the HR and HRV measures were found between the sleep stages, and the patterns of these changes were similar between both Scottish and NZ cows. Finally, machine learning models were developed using supervised learning methods to predict sleep stage from the HR and HRV measures as well as the surface EMG data recorded during PSG. The models were able to learn to recognize and accurately predict sleep stages compared with the PSG scoring. This research demonstrates that non-invasive alternatives can be used to identify sleep stages accurately in dairy cows compared with PSG. Further research is necessary with larger sample sizes and cows of various breeds and life stages; however, the success of the methods developed during this thesis demonstrates their suitability for the future measurement of sleep in cows and in the assessment of cow welfare

Structures of bacterial kynurenine formamidase reveal a crowded binuclear zinc catalytic site primed to generate a potent nucleophile

Tryptophan is an important precursor for chemical entities that ultimately support the biosynthesis of key metabolites. The second stage of tryptophan catabolism is catalysed by kynurenine formamidase, an enzyme that is different between eukaryotes and prokaryotes. In the present study, we characterize the catalytic properties and present the crystal structures of three bacterial kynurenine formamidases. The structures reveal a new amidase protein fold, a highly organized and distinctive binuclear Zn2+ catalytic centre in a confined, hydrophobic and relatively rigid active site. The structure of a complex with 2-aminoacetophenone delineates aspects of molecular recognition extending to the observation that the substrate itself may be conformationally restricted to assist binding in the confined space of the active site and for subsequent processing. The cations occupy a crowded environment, and, unlike most Zn2+ -dependent enzymes, there is little scope to increase co-ordination number during catalysis.We propose that the presence of a bridging water/hydroxide ligand in conjunction with the placement of an active site histidine supports a distinctive amidation mechanism.</p

ROLE OF THE GAMETE MEMBRANES IN FERTILIZATION IN SACCOGLOSSUS KOWALEVSKII (ENTEROPNEUSTA) : II. Zygote Formation by Gamete Membrane Fusion

An earlier paper showed that in Saccoglossus the acrosomal tubule makes contact with the egg plasma membrane. The present paper includes evidence that the sperm and egg plasma membranes fuse to establish the single continuous zygote membrane which, consequently, is a mosaic. Contrary to the general hypothesis of Tyler, pinocytosis or phagocytosis plays no role in zygote formation. Contact between the gametes is actually between two newly exposed surfaces: in the spermatozoon, the surface was formerly the interior of the acrosomal vesicle; in the egg, it was membrane previously covered by the egg envelopes. The concept that all the events of fertilization are mediated by a fertilizin-antifertilizin reaction seems an oversimplification of events actually observed: rather, the evidence indicates that a series of specific biochemical interactions probably would be involved. Gamete membrane fusion permits sperm periacrosomal material to meet the egg cytoplasm; if an activating substance exists in the spermatozoon it probably is periacrosomal rather than acrosomal in origin. The contents of the acrosome are expended in the process of delivering the sperm plasma membrane to the egg plasma membrane. After these membranes coalesce, the sperm nucleus and other internal sperm structures move into the egg cytoplasm

ROLE OF THE GAMETE MEMBRANES IN FERTILIZATION IN SACCOGLOSSUS KOWALEVSKII (ENTEROPNEUSTA) : I. The Acrosomal Region and Its Changes in Early Stages of Fertilization

Previous electron microscope studies of sperm-egg association in the annelid Hydroides revealed novel aspects with respect to the acrosomal region. To determine whether these aspects were unique, a comparable study was made of a species belonging to a widely separated phylum, Hemichordata. Osmium tetroxide-fixed polyspermic material of the enteropneust, Saccoglossus, was used. The acrosomal region includes the membrane-bounded acrosome, with its large acrosomal granule and shallow adnuclear invagination, and the periacrosomal material which surrounds the acrosome except at the apex; here, the acrosomal membrane lies very close to the enclosing sperm plasma membrane. After reaching the egg envelope, the spermatozoon is activated and undergoes a series of changes: the apex dehisces and around the resulting orifice the acrosomal and sperm plasma membranes form a continuous mosaic membrane. The acrosomal granule disappears. Within 7 seconds the invagination becomes the acrosomal tubule, spans the egg envelopes, and meets the egg plasma membrane. The rest of the acrosomal vesicle everts. The periacrosomal mass changes profoundly: part becomes a fibrous core (possibly equivalent to a perforatorium); part remains as a peripheral ring. The basic pattern of structure and sperm-egg association in Saccoglossus is the same as in Hydroides. Previous evidence from four other phyla as interpreted here also indicates conformity to this pattern. The major role of the acrosome is apparently to deliver the sperm plasma membrane to the egg plasma membrane

CHANGES IN THE SPERMATOZOON DURING FERTILIZATION IN HYDROIDES HEXAGONUS (ANNELIDA) : II. Incorporation with the Egg

This, the last of a series of three papers, deals with the final events which lead to the incorporation of the spermatozoon with the egg. The material used consisted of moderately polyspermic eggs of Hydroides hexagonus, osmium-fixed at various times up to five minutes after insemination. The first direct contact of sperm head with egg proper is by means of the acrosomal tubules. These deeply indent the egg plasma membrane, and consequently at the apex of the sperm head the surfaces of the two gametes become interdigitated. But at first the sperm and egg plasma membranes maintain their identity and a cross-section through the region of interdigitation shows these two membranes as a number of sets of two closely concentric rings. The egg plasma membrane rises to form a cone which starts to project into the hole which the spermatozoon earlier had produced in the vitelline membrane by means of lysis. But the cone does not literally engulf the sperm head. Instead, where they come into contact, sperm plasma membrane and egg plasma membrane fuse to form one continuous membranous sheet. At this juncture the two gametes have in effect become mutually incorporated and have formed a single fertilized cell with one continuous bounding membrane. At this time, at least, the membrane is a mosaic of mostly egg plasma membrane and a patch of sperm plasma membrane. The evidence indicates that the fusion of the two membranes results from vesiculation of the sperm and egg plasma membranes in the region at which they come to adjoin. Once this fusion of membranes is accomplished, the egg cytoplasm intrudes between the now common membrane and the internal sperm structures, such as the nucleus, and even extends into the flagellum; finally these sperm structures come to lie in the main body of the egg. The vesiculation suggested above appears possibly to resemble pinocytosis, with the difference that the vesicles are formed from the plasma membranes of two cells. At no time, however, is the sperm as a whole engulfed and brought to the interior of the egg within a large vesicle

FINE STRUCTURE OF THE SPERMATOZOON OF HYDROIDES HEXAGONUS (ANNELIDA), WITH SPECIAL REFERENCE TO THE ACROSOMAL REGION

This paper describes in some detail the structure of the acrosomal region of the spermatozoon of Hydroides as a basis for subsequent papers which will deal with the structural changes which this region undergoes during fertilization. The material was osmium-fixed and mild centrifugation was used to aggregate the spermatozoa from collection to final embedding. The studies concern also the acrosomal regions of frozen-thawed sperm prepared by a method which previously had yielded extracts with egg membrane lytic activity. The plasma membrane closely envelops four readily recognizable regions of the spermatozoon: acrosomal, nuclear, mitochondrial, and flagellar. The acrosome consists of an acrosomal vesicle which is bounded by a single continuous membrane, and its periphery is distinguishable into inner, intermediate, and outer zones. The inner and intermediate zones form a pocket into which the narrowed apex of the nucleus intrudes. Granular material adjoins the inner surface of the acrosomal membrane, and this material is characteristically different for each zone. Centrally, the acrosomal vesicle is spanned by an acrosomal granule: its base is at the inner zone and its apex at the outer zone. The apex of the acrosomal granule flares out and touches the acrosomal membrane over a limited area. In this limited area the adjoining granular material of the outer zone is lacking. The acrosomal membrane of the inner zone is invaginated into about fifteen short tubules. The acrosomal membrane of the outer zone is closely surrounded by the plasma membrane. At the apex of the acrosomal region a small apical vesicle is sandwiched between the plasma membrane and the acrosomal membrane. Numerous frozen-thawed specimens and occasional specimens not so treated show acrosomal regions at the apex of which there is a well defined opening or orifice. Around the rim or lip of this orifice plasma and acrosomal membranes may even be fused into a continuum. The evidence indicates that the apical vesicle and the parts of the plasma and acrosomal membranes which surround it constitute a lid, and the rim of this lid constitutes a natural "fracture line" or rim of dehiscence. Should fracture occur, the lid would be removed and the acrosomal vesicle would be open to the exterior

Egg Membrane Lytic Activity of Sperm Extract and its Significance in Relation to Sperm Entry in Hydroides hexagonus (Annelida)

Previous electron microscope studies indicated that the individual spermatozoön of Hydroides hexagonus forms a hole in the vitelline membrane by means of lysis. Other observations established that the hole is real, being visible in living material during sperm entry. During the present investigation sea water extracts from frozen-thawed sperm were tested for lytic effect on the membrane. In normal living eggs the membrane appears as a single thick envelope, but in electron micrographs of sections it is seen to consist of a narrow outer border layer, a wide principal or middle layer, and a narrow inner border layer. After immersion in sperm extract the outer border layer elevates but does not dissolve, the middle layer liquefies and disappears, and the inner border layer seems not to change. This is interpreted as lysis of the middle layer. The extract exerted the same effect on fertilized and unfertilized eggs. In electron micrographs the sections treated with extract greatly resemble that part of the membrane which has been penetrated by the individual spermatozoön. It is concluded that the individual spermatozoön, too, exerts a lytic effect. Together, the present and two earlier studies are considered clearly to demonstrate that in Hydroides the individual spermatozoön does indeed make an entry hole in the egg membrane by applying lytic material to that part of the membrane in its own vicinity

Formation of Sperm Entry Holes in the Vitelline Membrane of Hydroides hexagonus (Annelida) and Evidence of their Lytic Origin

Electron micrographs of inseminated eggs of Hydroides hexagonus previously had shown that in the immediate vicinity of the penetrating spermatozoön a small portion of the vitelline membrane regularly was absent, and it had been suggested that this area was a hole made by lytic activity of the individual spermatozoön during the course of its passage through the membrane. This deduction would receive support if it could be established that a sperm entry hole does form in living material. During the present study a hole repeatedly observed and photographed in the membrane of living eggs was found to arise as the spermatozoön penetrated the membrane. Gently compressed eggs formed exovates only through this hole. The holes, and exovates, were not found except at sperm entry sites. It was concluded that this hole is the counterpart of the area from which the membrane is absent in the electron micrographs cited above, and that the spermatozoön makes this hole. In an electron micrograph two spermatozoa which had penetrated the membrane at separate but closely neighboring points now occupy a single hole. It is argued that if each spermatozoön had displaced the membrane mechanically to make its hole, then there should be two holes, with a partition of membrane between them, but if each had eroded the membrane by applying lysin, a single hole should have formed as the eroded areas expanded and finally merged into one. The latter view agrees with the facts of the electron micrograph. It is concluded that lysis is the most probable means by which the individual spermatozoön makes its hole