The effect of consignment to broodmare sales on physiological stress measured by faecal glucocorticoid metabolites in pregnant thoroughbred mares

BACKGROUND: Validation of a method for the minimally-invasive measurement of physiological stress will help understanding of risk factors that may contribute to stress-associated events including recrudescence of Equid herpesvirus (EHV), which is anecdotally associated with sales consignment of pregnant Thoroughbred mares. In this study we compared two similar groups of late-gestation Thoroughbred broodmares on the same farm: a consigned Sales group (N = 8) and a non-consigned Control group (N = 6). The Sales mares were separated from their paddock companions and grouped prior to their preparation for, transport to, and return from the sales venue. Both groups were monitored by sampling at regular intervals from 5 days prior to until 14 days after the sales date (D0) to measure physiological stress in terms of changes in faecal glucocorticoid metabolite (FGM) concentrations, and for event-related viral recrudescence via daily body temperature measurements and periodic nasal swabs for PCR analysis for EHV-1 and −4 DNA. RESULTS: In both groups, FGM levels increased post-sales before returning to pre-sales levels. Specifically, FGM concentrations in the Sales mares were significantly higher on D + 3 and D + 10 than on D-4 and D-3 (F = 12.03, P < 0.0001, Post hoc: P = 0.0003 – 0.0008) and in the Control group FGM concentrations were higher on D + 10 than D-4 (F = 5.52, P = 0.004, Post hoc: P = 0.005). Interestingly, mean FGM levels in Control mares were significantly higher at 4 of the 5 sampling points (t = 5.64 – 2.25, p = 0.0001 – 0.044). Only one (Sales) mare showed PCR evidence of EHV-1 shedding. CONCLUSIONS : Using FGM to measure physiological stress was supported by the increases observed in all mares after Sales consignment, including those not consigned to the sale. Monitoring FGM levels therefore represents an appropriate, minimally-invasive method for future studies to assess the contribution of physiological stress to EHV recrudescence in horses transported to sales or equestrian events.The Equine Research Centre of the University of Pretoria funded the study.http://www.biomedcentral.com/bmcvetresam201

Quantitative Proteomic Analysis of Seminal Plasma, Sperm Membrane Proteins, and Seminal Extracellular Vesicles Suggests Vesicular Mechanisms Aid in the Removal and Addition of Proteins to the Ram Sperm Membrane

Quantitative proteomic studies are contributing greatly to the understanding of the spermatozoon through the provision of detailed information on the proteins spermatozoa acquire and shed in the acquisition of fertility. Extracellular vesicles (EVs) are thought to aid in the delivery of proteins to spermatozoa in the male reproductive tract. The aim of this study is to isolate, identify and quantify EV proteins isolated from ram seminal plasma. Ram sperm plasma membrane proteins are also isolated using nitrogen cavitation and identified to better understand the interplay of proteins between the sperm membrane and extracellular environment. The categorization of proteins enriched in the EV population according to their function revealed three main groupings: vesicle biogenesis, metabolism, and membrane adhesion and remodeling. The latter group contains many reproduction-specific proteins that show demonstrable links to sperm fertility. Many of these membrane-bound proteins show testicular expression and are shed from the sperm surface during epididymal maturation (e.g., testis expressed 101; TEX101 and lymphocyte Antigen 6 Family Member K; LY6K). Their association with seminal EVs suggests that EVs may not only deliver protein cargo to spermatozoa but also assist in the removal of proteins from the sperm membrane

Male Infertility: Shining a Light on Lipids and Lipid-Modulating Enzymes in the Male Germline

Despite the prevalence of male factor infertility, most cases are defined as idiopathic, thus limiting treatment options and driving increased rates of recourse to assisted reproductive technologies (ARTs). Regrettably, our current armory of ARTs does not constitute therapeutic treatments for male infertility, thus highlighting an urgent need for novel intervention strategies. In our attempts to fill this void, we have come to appreciate that the production of pathological levels of oxygen radicals within the male germline are a defining etiology of many idiopathic infertility cases. Indeed, an imbalance of reactive oxygen species can precipitate a cascade of deleterious sequelae, beginning with the peroxidation of membrane lipids and culminating in cellular dysfunction and death. Here, we shine light on the importance of lipid homeostasis, and the impact of lipid stress in the demise of the male germ cell. We also seek to highlight the utility of emerging lipidomic technologies to enhance our understanding of the diverse roles that lipids play in sperm function, and to identify biomarkers capable of tracking infertility in patient cohorts. Such information should improve our fundamental understanding of the mechanistic causes of male infertility and find application in the development of efficacious treatment options

The fate of porcine sperm CRISP2 from the perinuclear theca before and after in vitro fertilization

In a previous study, we reported that porcine sperm cysteine-rich secretory protein 2 (CRISP2) is localized in the post-acrosomal sheath-perinuclear theca (PT) as reduction-sensitive oligomers. In the current study, the decondensation and removal of CRISP2 was investigated during in vitro sperm capacitation, after both the induction of the acrosome reaction and in vitro fertilization. Confocal immunofluorescent imaging revealed that additional CRISP2 fluorescence appeared on the apical ridge and on the equatorial segment (EqS) of the sperm head following capacitation, likely due to cholesterol removal. After an ionophore A23187-induced acrosome reaction, CRISP2 immunofluorescence disappeared from the apical ridge and the EqS area partly not only owing to the removal of the acrosomal shroud vesicles, but to its presence in a subdomain of EqS. The fate of sperm head CRISP2 was further examined post-fertilization. In vitro matured porcine oocytes were co-incubated with boar sperm cells for 6-8 h and the zygotes were processed for CRISP2 immunofluorescent staining. Notably, decondensation of CRISP2, and thus of the sperm PT, occurred while the sperm nucleus was still fully condensed. CRISP2 was no longer detectable in fertilized oocytes in which sperm nuclear decondensation and paternal pronucleus formation were apparent. This rapid dispersal of CRISP2 in the PT is likely regulated by redox reactions for which its cysteine-rich domain is sensitive. Reduction of disulfide bridges within CRISP2 oligomers may be instrumental for PT dispersal and elimination

Heat stress in a temperate climate leads to adapted sensor-based behavioral patterns of dairy cows

Most research on heat stress has focused on (sub)tropical climates. The effects of higher ambient temperatures on the daily behavior of dairy cows in a maritime and temperate climate are less studied. With this retrospective observational study, we address that gap by associating the daily time budgets of dairy cows in the Netherlands with daily temperature and temperature-humidity index (THI) variables. During a period of 4 years, cows on 8 commercial dairy farms in the Netherlands were equipped with neck and leg sensors to collect data from 4,345 cow lactations regarding their daily time budget. The time spent eating, ruminating, lying, standing, and walking was recorded. Individual cow data were divided into 3 data sets: (1) lactating cows from 5 farms with a conventional milking system (CMS) and pasture access, (2) lactating cows from 3 farms with an automatic milking system (AMS) without pasture access, and (3) dry cows from all 8 farms. Hourly environment temperature and relative humidity data from the nearest weather station of the Dutch National Weather Service was used for THI calculation for each farm. Based on heat stress thresholds from previous studies, daily mean temperatures were grouped into 7 categories: 0 = (<0°C), 1 = (0–12°C, reference category), 2 = (12–16°C), 3 = (16–20°C), 4 = (20–24°C), 5 = (24–28°C), and 6 = (≥28°C). Temperature-humidity index values were grouped as follows: 0 = (THI <30), 1 = (THI 30–56, reference category), 2 = (THI 56–60), 3 = (THI 60–64), 4 = (THI 64–68), 5 = (THI 68–72) and 6 = (THI ≥72). To associate daily mean temperature and THI with sensor-based behavioral parameters of dry cows and of lactating cows from AMS and CMS farms, we used generalized linear mixed models. In addition, associations between sensor data and other climate variables, such as daily maximum and minimum temperature, and THI were analyzed. On the warmest days, eating time decreased in the CMS group by 92 min/d, in the AMS group by 87 min/d, and in the dry group by 75 min/d compared with the reference category. Lying time decreased in the CMS group by 36 min/d, in the AMS group by 56 min/d, and in the dry group by 33 min/d. Adaptation to daily temperature and THI was already noticeable from a mean temperature of 12°C or a mean THI of 56 or above, when dairy cows started spending less time lying and eating and spent more time standing. Further, rumination time decreased, although only in dry cows and cows on AMS farms. With higher values for daily mean THI and temperature, walking time decreased as well. These patterns were very similar for temperature and THI variables. These results show that dairy cows in temperate climates begin to adapt their behavior at a relatively low mean environmental temperature or THI. In the temperate maritime climate of the Netherlands, our results indicate that daily mean temperature suffices to study the effects of behavioral adaptation to heat stress in dairy cows

Reliability of pelvimetry is affected by observer experience but not by breed and sex: a cross‐sectional study in beef cattle

Pelvis size plays an important role to prevent dystocia in cattle caused by the foeto‐maternal disproportion in commonly primiparous females. The reproducibility and repeatability are 2 important aspects for the reliability of the measurements to use in the selection of cattle for culling. Pelvic measures were taken with a Rice pelvimeter from 224 young cattle (180 females and 44 males) of 4 beef breeds in South Africa. One experienced and 2 inexperienced observers each measured pelvic height and width twice. The proportion measurements with a maximum difference of 0.5 cm within animal compared to the first measurement by the experienced observer is around 80% and by the unexperienced observers around 50% for pelvic height and around 60% for pelvic width. Breed and sex don’t affect the reliability of pelvimetry by an experienced observer. Under‐ and overestimation of pelvis size was observed in inexperienced observers which seems to be unrelated to breed and sex

The effects of cow introductions on milk production and behaviour of the herd measured with sensors

This research paper addresses the hypothesis that cow introductions in dairy herds affect milk production and behaviour of animals already in the herd. In dairy farms, cows are commonly regrouped or moved. Negative effects of regroupings on the introduced animals are reported in other studies. However, little is known about the effects on lactating cows in the herd. In this research a herd of 53 lactating dairy cows was divided into two groups in a cross-over design study. 25 cows were selected as focal cows for which continuous sensor data were collected. The treatment period consisted of replacing non-focal cows three times a week. Many potentially influencing factors were taken into account in the analysis. Replacement of cows in the treatment period indeed affected the focal animals. During the treatment period these cows showed increased walking and reduced rumination activity and produced less milk compared to the control period. Milk production per milking decreased in the treatment period up to 0.4 kg per milking on certain weekdays. Lying and standing behaviour were similar between the control and the treatment period. The current study suggests that cow introductions affect welfare and milk production of the cows already in the herd

HDL mediates reverse cholesterol transport from ram spermatozoa and induces hyperactivated motility

Reverse cholesterol transport or cholesterol efflux is part of an extensive plasma membrane remodeling process in spermatozoa that is imperative for fertilization. For ram spermatozoa, sheep serum is well known to support in vitro fertilization (IVF), but knowledge of its explicit role is limited. Though, it is postulated to elicit cholesterol efflux owing to the presence of high-density lipoproteins (HDLs) that interact with transmembrane cholesterol transporters, such as adenosinetriphosphate (ATP)-binding cassette transporter A1 (ABCA1) and scavenger receptor class B, type I (SR-BI). In this study, we report that both sheep serum and HDLs were able to elicit cholesterol efflux alone by up to 20-40% (as measured by the boron dipyrromethene (BODIPY)-cholesterol assay). Furthermore, when the antagonists glibenclamide and valspodar were used to inhibit the function of ABCA1 and SR-BI or ABCA1 alone, respectively, cholesterol efflux was only marginally reduced (8-15%). Nevertheless, it is likely that in ram spermatozoa, a specific facilitated pathway of cholesterol efflux is involved in the interaction between cholesterol acceptors and transporters. Interestingly, exposure to HDLs also induced hyperactivated motility, another critical event required for successful fertilization. Taken together, this study details the first report of the dual action of HDLs on ram spermatozoa, providing both an insight into the intricacy of events leading up to fertilization in vivo as well as demonstrating the possible application of HDL supplementation in media for IVF

Pressure measurement in the reticulum to detect different behaviors of healthy cows

The aim of the current study was to investigate the relation between reticulorumen contractions and monitored cow behaviors. A purpose-built pressure measuring device was used and shown to be capable of detecting the known contraction patterns in the reticulorumen of four rumen-fistulated cows. Reticular pressure data was used to build a random forest algorithm, a learning algorithm based on a combination of decision trees, to detect rumination and other cow behaviors. In addition, we developed a peak-detection algorithm for rumination based on visual inspection of patterns in reticular pressure. Cow behaviors, differentiated in ruminating, eating, drinking, sleeping and 'other', as scored from video observation, were used to develop and test the algorithms. The results demonstrated that rumination of a cow can be detected by measuring pressure differences in the reticulum using either the random forest algorithm or the peak-detection algorithm. The random forest algorithm showed very robust performances for detecting rumination with an accuracy of 0.98, a sensitivity of 0.95 and a specificity of 0.99. The peak-detection algorithm could detect rumination robustly, with an accuracy of 0.92, a sensitivity of 0.97 and a specificity of 0.90. In addition, we provide proof of principle that a random forest algorithm can also detect eating, drinking and sleeping behavior from the same data with performances above 0.90 for all measures. The measurement device used in this study needed rumen-fistulated cows, but the results indicate that behavior detection using algorithms based on only measurements in the reticulum is feasible. This is promising as it may allow future wireless sensor techniques in the reticulum to continuously monitor a range of important behaviors of cows

Initiation of X Chromosome Inactivation during Bovine Embryo Development

X-chromosome inactivation (XCI) is a developmental process that aims to equalize the dosage of X-linked gene products between XY males and XX females in eutherian mammals. In female mouse embryos, paternal XCI is initiated at the 4-cell stage; however, the X chromosome is reactivated in the inner cell mass cells of blastocysts, and random XCI is subsequently initiated in epiblast cells. However, recent findings show that the patterns of XCI are not conserved among mammals. In this study, we used quantitative RT-PCR and RNA in situ hybridization combined with immunofluorescence to investigate the pattern of XCI during bovine embryo development. Expression of XIST (X-inactive specific transcript) RNA was significantly upregulated at the morula stage. For the first time, we demonstrate that XIST accumulation in bovine embryos starts in nuclei of female morulae, but its colocalization with histone H3 lysine 27 trimethylation was first detected in day 7 blastocysts. Both in the inner cell mass and in putative epiblast precursors, we observed a proportion of cells with XIST RNA and H3K27me3 colocalization. Surprisingly, the onset of XCI did not lead to a global downregulation of X-linked genes, even in day 9 blastocysts. Together, our findings confirm that diverse patterns of XCI initiation exist among developing mammalian embryos