An Investigation of Equine Sperm Quality Following Cryopreservation at Low Sperm Concentration and Repeated Freeze-Thawing.

Stallion spermatozoa are typically cryopreserved at 200 to 300 million sperm/ml; however recent advances such as intracytoplasmic sperm injection (ICSI) requires only one spermatozoon, wasting many, after thawing a whole straw. Cryopreserving at concentrations less than the current standard or refreezing thawed spermatozoa could maximize the use of genetically valuable animals and reduce waste. This investigation aimed to identify if lowering the sperm concentration for cryopreservation affected post-thaw quality after one and two freeze-thaw cycles. Nine ejaculates were collected from three fertile, "good freezer" stallions (post-thaw motility ≥35%) for experiment 1. Each ejaculate was split into eight treatments: five, 10, 20, 50, 100, 200, 300, 400 million sperm/ml and cryopreserved. Post-thaw: motility, viability, acrosome integrity and oxidative stress were assessed. Experiment 2, straws from experiment 1 (300 million sperm/ml) were thawed, diluted to 20 million sperm/ml or left undiluted (control) and refrozen. Post-thaw motility and viability were assessed. In experiment 1 sperm concentration did not affect post-thaw total motility (TM), progressive motility (PM) or viability at 50 to 400 million sperm/ml (P > .05). Whilst sperm concentrations of five to 20 million/ml did differ (post-thaw TM and PM). Both refreezing and reducing spermatozoa concentration, decreased TM, PM and viability (P < .05) after two freeze-thaw cycles. These results suggest cryopreserving at sperm concentrations as low as 50 million/ml maintains spermatozoa quality in good freezer stallions. Spermatozoa maintained some motility and viability when initially cryopreserved at 20 million sperm/ml and after two freeze-thaw cycles but research should investigate more optimal conditions.

Assessing the Accuracy of Leg Mounted Sensors for Recording Dairy Cow Behavioural Activity at Pasture, in Cubicle Housing and a Straw Yard.

The accuracy of CowAlert IceQube sensors (IceRobotics Ltd., Edinburgh, UK) for recording lying duration, standing and lying transitions and number of steps when dairy cows where at pasture, in cubicle housing and in a straw yard, was investigated. Holstein Friesian cows at Harper Adams University, UK were fitted with IceQube sensors; one on the back left (BL) leg and one on the front left (FL) leg. Cows at pasture (n = 48), in cubicle housing (n = 46) and in a straw yard (n = 45) were visually observed. Data were analysed in two stages: (1) an initial exploratory phase determined the correlation level between sensor measurements andvisual observations. Subsequently, (2) a mixed effects modelling framework was used to check whether sensors provide significantly different measures of cow's activities compared to the observations. Results indicate that lying and standing times are similar between the observed and recorded times, in all three locations. In terms of sensor placement, significant differences were found between the number of steps recorded between BL and FL on straw and pasture, but all other activities were similar, in each location. The accuracy of CowAlert IceQube sensors on the BL leg gives them the potential to be used as lifelong sensors.

Stakeholder Perceptions of Disease Management for Dairy Calves: "It's Just Little Things That Make Such a Big Difference".

Calf morbidity and mortality rates are often high in dairy herds, raising animal welfare concerns and negatively affecting farm economic efficiency and future performance. Disease prevention is critical to maintain calves in good health, but interventions are dependent upon the persons conducting them. This paper explores the perceptions of farmers, farm workers, veterinarians, and other advisors on the management of calfhood disease on dairy farms in England. Participants were recruited using purposive and "snowball" sampling, resulting in 40 in-depth, semi-structured interviews-26 with dairy farmers and 14 with advisors. Interviews were recorded, transcribed, and thematically coded. Three major themes were derived on the basis of interview data: disease occurrence and treatments, management of calf environment, and the role of stockmanship and perceived control. Respiratory and gastrointestinal diseases in calves were those reported to be most problematic on dairy farms. Limited time and financial resources caused some farmers and advisors to experience a perceived inability to control calf health without antimicrobial treatments. Overall, the findings emphasise the importance of human influences on calf health and disease in the context of influencing the interactions among the host, pathogens, and the environment. Further research should investigate what "attention to detail" means within different farm contexts and practices, as this was believed to be important in the promotion of better husbandry standards and health. We recommend the use of supportive knowledge exchange processes, including facilitation, to empower farmers to promote continuous improvement in calf health.

Co-Produced Care in Veterinary Services: A Qualitative Study of UK Stakeholders' Perspectives.

Changes in client behaviour and expectations, and a dynamic business landscape, amplify the already complex nature of veterinary and animal health service provision. Drawing on prior experiences, veterinary clients increasingly pursue enhanced involvement in services and have expectations of relationship-centred care. Co-production as a conceptualisation of reciprocity in service provision is a fundamental offering in the services sector, including human medicine, yet the role of co-production in veterinary services has been minimally explored. Utilising a service satisfaction framework, semi-structured interviews (n = 13) were completed with three veterinary stakeholder groups, veterinarians, allied animal health practitioners, and veterinary clients. Interview transcript data were subject to the qualitative data analysis techniques, thematic analysis and grounded theory, to explore relationship-centred care and subsequently conceptualise co-production service for the sector. Six latent dimensions of service were emergent, defined as: empathy, bespoke care, professional integrity, value for money, confident relationships, and accessibility. The dimensions strongly advocate wider sector adoption of a co-produced service, and a contextualised co-production framework is presented. Pragmatic challenges associated with integration of active veterinary clients in a practitioner-client partnership are evident. However, adopting a people-centric approach to veterinary services and partnerships with clients can confer the advantages of improved client satisfaction, enhanced treatment adherence and outcomes, and business sustainability.

Appropriate Dairy Calf Feeding from Birth to Weaning: "It's an Investment for the Future".

Dairy calves must be fed appropriately to meet their nutritional needs, supporting optimal growth and development to achieve the recommended target age at first calving (AFC) of 24 months. Traditional restricted milk feeding practices suppress growth, contribute to negative welfare states and may result in malnutrition and immunosuppression. Despite more recent recommendations to increase milk allowances for pre-weaned calves, restricted feeding remains a common practice. This study explored the rationales behind the calf feeding protocols used by dairy farmers in England. Forty qualitative interviews (26 farmers, 14 advisors) were conducted between May 2016 and June 2017, transcribed in full, then coded into themes. Results indicate that a variety of calf feeding regimes are used on farms, largely determined by farmers' attitudes regarding ease of management and the wellbeing of calves. Advisors were concerned about widespread underfeeding of calves, which may be partially due to insufficiently clear recommendations for calf milk replacer (CMR) feeding rates. There was also evidence of uncertainty regarding best practices for weaning calves. Collaboration between academic research and industry is essential to establish a consensus on calf feeding standards which support physiological function, facilitate weaning, support growth targets and ensure calf health and welfare is protected.

Cows with paratuberculosis (Johne's disease) alter their lying behavior around peak lactation.

Paratuberculosis or Johne's disease (JD) is a fatal chronic enteritis that causes detrimental effects on production and health and significantly reduces the welfare of cattle. Control of JD is highly desirable, but single milk ELISA testing may not be sensitive enough to identify all affected animals, particularly in the early stages of the disease. The objective of this study was to compare the activity of JD-positive (JD5) to JD-negative (JD0) cows from calving until wk 20 of lactation. The study was conducted at Harper Adams University, United Kingdom, using 42 multiparous [3.1 ± 0.22 (mean ± standard error of the mean); range: 2-7 lactations] Holstein Friesian cows, fitted with an IceQube accelerometer (IceRobotics Ltd., Edinburgh, UK) on the back left leg. The sensors recorded data on lying and standing time, steps, and motion index with a granularity of 15 min. In addition, start and stop times for lying bouts, and exact lying bout durations were recorded, which permits calculation of the number of lying bouts. Every 3 mo the cows were milk sampled and subsequently tested for JD using an ELISA. Cows in the infection group JD0 were classed as JD negative and cows in the infection group JD5 were classed as JD positive. Johne's-positive cows [JD5; n = 21 (repeat ELISA positive)] were matched to negative cows [JD0; n = 21 (repeat ELISA negative)] based on lactation number and age. Around peak lactation we found differences in lying behavior. The JD5 cows spend less time lying/d during wk 7 to 11 of lactation. The largest difference observed was around wk 8 of lactation, with JD5 cows spending, on average, 2 h/d less time lying down than JD0 cows (9.3 ± 0.33 vs. 11.3 ± 0.61 h/d, respectively). The JD5 cows also had fewer lying bouts per day from wk 7 to 15 of lactation (excluding wk 13), and during wk 11 and 12 average lying bout duration was longer for JD5 cows compared with JD0 cows. No differences were observed in steps per day, milk yield, BCS, and mobility score between JD5 and JD0 cows from calving to wk 20 of lactation. As far as we are aware, this is the first study to show changes in activity of JD-positive cows. The results show that activity data from leg-mounted accelerometers has the potential to help identify JD-positive cows, although more research is required.

Fatty acid profile of milk for determining reproductive status in lactating Holstein Friesian cows.

Large percentages of dairy cows do not express behavioural signs of oestrus. Faecal and urine fatty acid concentrations increase during oestrus. The objective of the present study was to determine the milk FA profile of dairy cows during the oestrous and dioestrous periods and the relationship with behavioural signs on the day of oestrus. The activity of 32 Holstein Friesian cows was measured continuously using GEA Rescounter ll pedometers (GEA Farm Technologies, Düsseldorf, Germany) and IceQubes (IceRobotics Ltd., Edinburgh, UK). Milk samples were collected on the day of oestrus and on day 14 of the subsequent oestrous cycle and analysed for FA concentration using gas chromatography (GC) and milk composition was also determined. All cows were artificially inseminated within 12 h of the onset of oestrus. On the day of oestrus, the concentration of acetic acid (P < 0.001), valeric acid (P = 0.016), caproic acid (P < 0.001) and myristoleic (P = 0.035) were greater in milk compared to day 14. On day 14 milk arachidonic acid concentration, however, was greater (P = 0.004) compared to the day of oestrus. Also, on day 14 arachidonic acid concentration was greater (P = 0.002) in non-pregnant compared to pregnant cows. In conclusion, the results of this study indicate there are changes in the concentrations of some milk FA during oestrus and dioestrus in lactating dairy cows.

Ovarian expression of insulin-like peptide 3 (INSL3) and its receptor (RXFP2) during development of bovine antral follicles and corpora lutea and measurement of circulating INSL3 levels during synchronized estrous cycles.

Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary, but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna cell (TIC) and granulosa cell compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than granulosa cell and increased progressively during follicle maturation with INSL3 peaking in large (11-18 mm) estrogen-active follicles and RXFP2 peaking in 9- to 10-mm follicles before declining in larger (11-18 mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly autocrine/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17ß followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant preovulatory follicle, is detectable in peripheral blood of cattle, and expression is down-regulated during luteinization induced by the preovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, while raising doubts about its potential contribution to CL function.

Association between ovarian follicle development and pregnancy rates in dairy cows undergoing spontaneous oestrous cycles.

Ovarian follicle development continues in a wave-like manner during the bovine oestrous cycle giving rise to variation in the duration of ovulatory follicle development. The objectives of the present study were to determine whether a relationship exists between the duration of ovulatory follicle development and pregnancy rates following artificial insemination (AI) in dairy cows undergoing spontaneous oestrous cycles, and to identify factors influencing follicle turnover and pregnancy rate and the relationship between these two variables. Follicle development was monitored by daily transrectal ultrasonography from 10 days after oestrus until the subsequent oestrus in 158 lactating dairy cows. The cows were artificially inseminated following the second observed oestrus and pregnancy was diagnosed 35 days later. The predominant pattern of follicle development was two follicle waves (74.7%) with three follicle waves in 22.1% of oestrous cycles and four or more follicle waves in 3.2% of oestrous cycles. The interval from ovulatory follicle emergence to oestrus (EOI) was 3 days longer (P < 0.0001) in cows with two follicle waves than in those with three waves. Ovulatory follicles from two-wave oestrous cycles grew more slowly but were approximately 2 mm larger (P < 0.0001) on the day of oestrus. Twin ovulations were observed in 14.2% of oestrous cycles and occurred more frequently (P < 0.001) in three-wave oestrous cycles; consequently EOI was shorter in cows with twin ovulations. Overall, 57.0% of the cows were diagnosed pregnant 35 days after AI. Linear logistic regression analysis revealed an inverse relationship between EOI and the proportion of cows diagnosed pregnant, among all cows (n = 158; P < 0.01) and amongst those with single ovulations (n = 145; P < 0.05). Mean EOI was approximately 1 day shorter (P < 0.01) in cows that became pregnant than in non-pregnant cows; however, pregnancy rates did not differ significantly among cows with different patterns of follicle development. These findings confirm and extend previous observations in pharmacologically manipulated cattle and show, for the first time, that in dairy cows undergoing spontaneous oestrous cycles, natural variation in the duration of post-emergence ovulatory follicle development has a significant effect on pregnancy rate, presumably reflecting variation in oocyte developmental competence.