Semen is one of a few naturally occurring monocellular suspensions, and therefore sperm function analysis by flow cytometry and utilization of fluorochromes is an ideal technique for high throughput, multiparametric, and accurate analysis. Sperm quality assessment often includes measures of cellular integrity and potential capability of spermatozoa to fertilize and stages of capacitation. When deciding on and setting up a sperm assay using flow cytometry, there are several factors to consider which affect the assay and the measured output. These include but are not limited to instrument type and settings and sample preparation. The classic sperm assays which use flow cytometry are sperm viability, acrosomal integrity, membrane stability, and mitochondrial status. Here, we describe principles and procedures to set up and develop these sperm function analyses using traditional flow cytometry.

SWATH is a data acquisition strategy acclaimed for generating quantitatively accurate and consistent measurements of proteins across multiple samples. Its utility for proteomics studies in nonlaboratory animals, however, is currently compromised by the lack of sufficiently comprehensive and reliable public libraries, either experimental or predicted, and relevant platforms that support their sharing and utilization in an intuitive manner. Here we describe the development of the Veterinary Proteome Browser, VPBrowse ( http://browser.proteo.cloud/ ), an on-line platform for genome-based representation of the Bos taurus proteome, which is equipped with an interactive database and tools for searching, visualization, and building quantitative mass spectrometry assays. In its current version (VPBrowse 1.0), it contains high-quality fragmentation spectra acquired on QToF instrument for over 36,000 proteotypic peptides, the experimental evidence for over 10,000 proteins. Data can be downloaded in different formats to enable analysis using popular software packages for SWATH data processing whilst normalization to iRT scale ensures compatibility with diverse chromatography systems. When applied to published blood plasma dataset from the biomarker discovery study, the resource supported label-free quantification of additional proteins not reported by the authors previously including PSMA4, a tissue leakage protein and a promising candidate biomarker of animal's response to dehorning-related injury.

Mass spectrometry-based plasma proteomics offers a major advance for biomarker discovery in the veterinary field, which has traditionally been limited to quantification of a small number of proteins using biochemical assays. The development of foundational data and tools related to sequential window acquisition of all theoretical mass spectra (SWATH)-mass spectrometry has allowed for quantitative profiling of a significant number of plasma proteins in humans and several animal species. Enabling SWATH in dogs enhances human biomedical research as a model species, and significantly improves diagnostic and disease monitoring capability. In this study, a comprehensive peptide spectral library specific to canine plasma proteome was developed and evaluated using SWATH for protein quantification in non-depleted dog plasma. Specifically, plasma samples were subjected to various orthogonal fractionation and digestion techniques, and peptide fragmentation data corresponding to over 420 proteins was collected. Subsequently, a SWATH-based assay was introduced that leveraged the developed resource and that enabled reproducible quantification of 400 proteins in non-depleted plasma samples corresponding to various disease conditions. The ability to profile the abundance of such a significant number of plasma proteins using a single method in dogs has the potential to accelerate biomarker discovery studies in this species.

Pain assessment in farm animals has primarily relied on a combination of behavioral and physiological responses, although these are relatively subjective and difficult to quantify. It is essential to develop more effective biomarkers of pain in production animals since they are frequently exposed to routine surgical husbandry procedures. More effective biomarkers of pain would improve welfare, limit the loss of productivity associated with pain and permit better assessment of analgesics. This study aimed to investigate the use of a modern mass spectrometry data independent acquisition strategy, termed Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS), to detect candidate protein biomarkers that are known to associate with nociceptive and inflammatory processes in cattle, which could then be used to assess the efficacy of potential analgesics. Calves were randomly divided into two groups that were either surgically dehorned or subjected to restraint stress, without provision of anaesthesia or analgesia in accordance with current industry standards. Samples were analysed before and after dehorning at multiple timepoints. Significant changes in protein concentrations were detected predominantly at 24 and 96 h following dehorning, including kininogens, proteins associated with the coagulation and complement cascades and serine protease inhibitors. Gene ontology analysis revealed that the identified candidate biomarkers were associated with stress, wound healing, immune response, blood coagulation and the inflammatory and acute phase responses, which could be expected following surgical damage to tissues, but can now be more objectively assessed. These results offer more definitive and quantitative monitoring of response to tissue injury induced pain and inflammation.

Dogs undergo various surgical procedures such as castration, ovariohysterectomy, and other orthopedic procedures, which are known to cause inflammation and pain. Non-steroidal anti-inflammatory drugs (NSAIDs) are very effective analgesics for alleviating postoperative pain in veterinary medicine. Ketoprofen is currently approved in Australia and the United States for treating different painful conditions in dogs. This study evaluated the pharmacokinetic parameters of ketoprofen after intravenous (IV) and transdermal (TD) administration in healthy dogs. A novel transdermal ketoprofen (TDK) formulation containing 20% ketoprofen, dissolved in a combination of 45:45% isopropanol and Transcutol, along with 10% eucalyptus oil, was developed and evaluated for in vitro dermal permeation using Franz diffusion cells. A crossover study was then conducted to determine the pharmacokinetic parameters of the formulation in six dogs following IV ketoprofen (1 mg/kg) and TDK (10 mg/kg) administration. A liquid chromatography–mass spectrometry (LC-M/MS) method was used to measure plasma concentrations of ketoprofen over time, and a non-compartmental analysis determined the pharmacokinetic parameters. The mean terminal elimination half-life (T½ h), AUC0-t (µg·h/mL), and mean residence time (MRT, h) between IV and TDK groups were 4.69 ± 1.33 and 25.77 ± 22.15 h, 15.75 ± 7.72 and 8.13 ± 4.28 µg·h/mL, and 4.86 ± 1.81 and 41.63 ± 32.33 h, respectively. The calculated bioavailability (F%) was ~7%, with a lag time of 30 min to achieve effective plasma concentrations after the application of TDK.

Assessment of pain responses and inflammation during animal surgery is difficult because traditional methods, such as visual analogue scores, are not applicable while under anaesthesia. Acute phase proteins (APPs), such as C-reactive protein and haptoglobin, that are typically monitored in veterinary research, do not show a significant change until at least 2 h post-surgery and therefore, immediate pathophysiological changes are uncertain. The current study used sequential window acquisition of all theoretical mass spectra (SWATH-MS) to investigate plasma proteome changes that occur immediately following surgery in dogs and also to assess the efficacy of a novel transdermal ketoprofen (TK) formulation. Castration was chosen as surgical model in this study. The procedure was performed on twelve dogs (n = 6 in two groups) and blood samples were collected at 0 h, 1 and 2 h after surgery for proteomic analysis. Following surgery, there was a general downregulation of proteins, including complement C- 3, complement factor B, complement factor D, transthyretin, and proteins associated with lipid, cholesterol, and glucose metabolisms, reflecting the systemic response to surgical trauma. Many of these changes were diminished in the transdermal group (TD) since ketoprofen, a non-steroidal anti-inflammatory drug (NSAID), inhibits prostanoids and the associated chemotactic neutrophil migration to site of tissue injury. Significance: SWATH-MS Proteomic analysis revealed significant changes in plasma proteins, predominantly involved in early acute phase and inflammatory response at 1 & 2 h after surgery in castrated dogs. Pre-operative application of transdermal ketoprofen formulation had reduced the systemic immune response, which was confirmed by negligible alteration of proteins in transdermal treated group. A key outcome of this experiment was studying the efficacy of a novel transdermal NSAID formulation in dogs.

Ketoprofen is registered in many countries for injectable administration in cattle. Because it is soluble in a wide range of excipients, development of a novel transdermal (TD) ketoprofen formulation was pursued to provide a convenient and pain-free route of administration in cattle. One hundred and six excipient combinations were screened using in vitro techniques (Franz diffusion cells), with a 20%(w/v) ketoprofen formulation dissolved in a combination of 45%:45%(v/v) ethanol and isopropyl myristate (IPM) and 10%(v/v) eucalyptus oil achieving maximal penetration of ketoprofen through bovine skin. A bioavailability study was then conducted using a randomized cross-over design (n = 12), including IV, IM (both 3 mg/kg) and TD (10 mg/kg) ketoprofen formulations administered with a one-week washout period between administrations. The IV and IM formulation pharmacokinetic results were as expected. The CMAX , Tmax and AUC0-Last were significantly higher (arithmetic mean ± SD) after TD administration (20.0 ± 6.5 μg/ml, 115 ± 17 min and 3940 ± 1324 μg*min/ml, respectively), compared to IM (11.0 ± 4.0 μg/ml, 74 ± 43 min and 2376 ± 738 μg*min/ml, respectively), although there were no significant differences for T½β . However, dose corrected values CMAX and AUCinf were significantly higher for IM compared to TD. The arithmetic mean bioavailability (F) of the transdermal formulation was 50%. The plasma concentration of the TD formulation at a dose of 10 mg/kg was similar to the IM formulation at 3 mg/kg by 30 min post-dosing with an arithmetic mean ± SD of 7.97 ± 4.38 vs. 8.02 ± 3.55 μg/ml, respectively. The TD formulation was generally well tolerated by cattle, although some local irritation along the site of application was noted after 12 h of exposure during the bioavailability study. Results indicate that this novel TD formulation provides a substantial improvement in administration convenience, may improve animal welfare and end-user safety through needle-free administration, and achieves similar plasma pharmacokinetics to the IM product when administered at 10 mg/kg.

The collection of blood plasma is minimally invasive, and the fluid is a rich source of proteins for biomarker studies in both humans and animals. Plasma protein analysis by mass spectrometry (MS) can be challenging, though modern data acquisition strategies, such as sequential window acquisition of all theoretical fragment ion spectra (SWATH), enable reproducible quantitation of hundreds of proteins in non-depleted plasma from humans and laboratory model animals. Although there is strong potential to enhance veterinary and translational research, SWATH-based plasma proteomics in non-laboratory animals is virtually non-existent. One limitation to date is the lack of comprehensively annotated genomes to aid protein identification. The current study established plasma peptide spectral repositories for sheep and cattle that enabled quantification of over 200 proteins in non-depleted plasma using SWATH approach. Moreover, bioinformatics pipeline was developed to leverage inter-species homologies to enhance the depth of baseline libraries and plasma protein quantification in bovids. Finally, the practical utility of using bovid libraries for SWATH data extraction in taxonomically related nondomestic ungulate species (giraffe) has been demonstrated. Significance: Ability to quickly generate comprehensive spectral libraries is limiting the applicability of dataindependent acquisition, such as SWATH, to study proteomes of non-laboratory animals. We describe an approach to obtain relatively shallow foundational plasma repositories from domestic ruminants and employ homology searches to increase the depth of data, which we subsequently extend to unsequenced ungulates using SWATH method. When applied to cross-species proteomics, the number of proteins quantified by our approach far exceeds what is traditionally used in plasma protein tests.

Endometrial infections are a common cause of reproductive loss in cattle. Accurate diagnosis is important to reduce the economic losses caused by endometritis. A range of sampling procedures have been developed which enable collection of endometrial tissue or luminal cells or uterine fluid. However, as these are all invasive procedures, there is a risk that sampling around the time of breeding may adversely affect subsequent pregnancy rate. This systematic review compared the pregnancy rates (PR) of cattle which underwent uterine lavage (UL), cotton swab (CS), cytobrush (CB), cytotape (CT), or endometrial biopsy (EB) sampling procedures with those that were not sampled. Using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) protocol, relevant databases, including Pubmed, Web of Science, CAB Abstracts, VetMed Resource–Ruminants, and Scopus, were searched. The outcome measured was the pregnancy rate after the collection of endometrial sample(s). Seven studies, involving a total of 3693 cows, fulfilled the inclusion criteria for the systematic review and allowed the comparison of PR between sampled (n = 1254) and non-sampled cows (n = 2409). The results of the systematic review showed that endometrial sampling procedures can be performed before breeding or shortly after insemination without adversely affecting pregnancy rates in cattle. However, further studies are needed to validate this information.

Environmental temperature has effects on sperm quality with differences in susceptibility between cattle subspecies and breeds, but very little is known about the seminal plasma protein (SPP) changes resulting from testicular heat stress. Scrotal insulation (SI) for 48 hr was applied to Brahman (Bos indicus) bulls. Semen was collected at 3-day intervals from before, until 74 days post-SI. The changes in sperm morphology and motility following SI were comparable to previously reported and differences were detected in measures of sperm chromatin conformation as early as 8 days post-SI. New proteins spots, in the SPP two-dimensional (2-D) gels, were apparent when comparing pre-SI with 74 days post-SI, and SPP identified as associated with mechanisms of cellular repair and protection. Similar trends between 2-D gel and Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) data was observed, with SWATH-MS able to quantify individual SPP that otherwise were not resolved on 2-D gel. The SPP assessment at peak sperm damage (21-24 days) showed a significant difference in 29 SPP (adjusted p < .05), and identified six proteins with change in abundance in the SI group. In conclusion both spermatozoa and SPP composition of bulls are susceptible to temperature change incurred by SI, and SPP markers for testicular heat insults may be detected.

There is a critical need to ensure that all cattle undergoing surgical husbandry procedures are provided effective pain relief. Non-steroidal anti-inflammatory drugs (NSAIDs) are most commonly used, and typically are administered by intramuscular (IM) injection. However, administration of NSAIDs via this route to large numbers of cattle which are handled only once or twice a year, typical of many rangeland beef production systems, presents significant occupational health and safety and mis-administration risks. To address this, a novel transdermal (TD) formulation of ketoprofen was developed, and its efficacy assessed in a study of 36 Holstein–Friesian calves which were assigned to a placebo (n = 10), a TD ketoprofen (n = 10), an IM ketoprofen (n = 10) and sham dehorned group (n = 6). TD ketoprofen significantly reduced plasma cortisol concentrations between 1 to 4 h after dehorning compared to placebo treated calves, with concentrations at 2 and 4 h being very similar to those for sham dehorned calves. The expected log count of positively associated pain variables (ear flick, tail wag, ruminating, head shake, lying down, grooming and neck extending) in the TD group was reduced by 42%, compared to placebo calves, with an overall significant (p < 0.05) treatment effect. The IM group exhibited similar responses and both TD and IM cattle had a higher BW gain at 2 and 5 (p < 0.05) weeks post-dehorning, compared to placebo. This study has shown that TD administered ketoprofen was at least as effective as IM to control pain associated with dehorning and facilitates the administration of analgesic drugs prior to the surgical husbandry procedures being performed.

Background Twenty-five phenotypes were measured as indicators of bull fertility (1099 Brahman and 1719 Tropical Composite bulls). Measurements included sperm morphology, scrotal circumference, and sperm chromatin phenotypes such as DNA fragmentation and protamine deficiency. We estimated the heritability of these phenotypes and carried out genome-wide association studies (GWAS) within breed, using the bovine high-density chip, to detect quantitative trait loci (QTL). Results Our analyses suggested that both sperm DNA fragmentation and sperm protamine deficiency are heritable (h 2 from 0.10 to 0.22). To confirm these first estimates of heritability, further studies on sperm chromatin traits, with larger datasets are necessary. Our GWAS identified 12 QTL for bull fertility traits, based on at least five polymorphisms (P < 10 −8 ) for each QTL. Five QTL were identified in Brahman and another seven in Tropical Composite bulls. Most of the significant polymorphisms detected in both breeds and nine of the 12 QTL were on chromosome X. The QTL were breed-specific, but for some traits, a closer inspection of the GWAS results revealed suggestive single nucleotide polymorphism (SNP) associations (P < 10 −7 ) in both breeds. For example, the QTL for inhibin level in Braham could be relevant to Tropical Composites too (many polymorphisms reached P < 10 −7 in the same region). The QTL for sperm midpiece morphological abnormalities on chromosome X (QTL peak at 4.92 Mb, P < 10 −17 ) is an example of a breed-specific QTL, supported by 143 significant SNPs (P < 10 −8 ) in Brahman, but absent in Tropical Composites. Our GWAS results add evidence to the mammalian specialization of the X chromosome, which during evolution has accumulated genes linked to spermatogenesis. Some of the polymorphisms on chromosome X were associated to more than one genetically correlated trait (correlations ranged from 0.33 to 0.51). Correlations and shared polymorphism associations support the hypothesis that these phenotypes share the same underlying cause, i.e. defective spermatogenesis. Conclusions Genetic improvement for bull fertility is possible through genomic selection, which is likely more accurate if the QTL on chromosome X are considered in the predictions. Polymorphisms associated with male fertility accumulate on this chromosome in cattle, as in humans and mice, suggesting its specialization.

In the quest for predicting fertility of an individual, enhancing semen handling, dilution and storage protocols, and understanding the impact of environment and, andrologists have changed their approaches to semen analysis. The technologies used today are fast developing and readily implemented in research. Semen is one of a few naturally occurring monocellular suspensions, so sperm function analysis by flow cytometry (FC) and utilization of fluorochromes is an ideal technique for high throughput, objective and accurate analysis. The complementary use of microscopical assessments by Computer-Assisted Semen Analysis (CASA), where sperm cell parameters can be objectively assessed is equally important. The objectivity and repeatability of these techniques have driven research on the function, identification of heterogeneity and fertility of the ejaculate. The wealth of knowledge obtained from the application of these powerful methods has changed our view of the spermatozoon. Although there is some application of these methods in the industry producing boar semen for artificial insemination (AI) and to eliminate sires of sub-standard semen quality, uptake of advanced methods is still slow. Instruments are becoming cheaper and technically more user friendly. Standardization of methodology and optimization of instrument settings is important for full implementation of these systems, including comparison between labs. This review provides an update on two technologies: flow cytometry and CASA for objective analysis of boar semen quality.

Reproductive procedures involving uterine manipulation (UM), such as transcervical embryo transfer (TET) or the collection of an endometrial biopsy (EB) may cause trauma to the endometrium resulting in release of endogenous prostaglandin F2α (PGF2α) causing premature regression of the corpus luteum (CL). This study aimed to evaluate the effect of UM and EB on corpus luteum lifespan in tropically adapted beef heifers. In Experiment 1, pre-synchronised Droughtmaster heifers (n = 9; 25.8 ± 0.4 m, live weight 391 ± 21.5 kg) were randomly selected either at Day 4 (UM4 = 5) or at Day 7 (UM7 = 4) post-oestrus for transrectal uterine manipulation and transcervical passage of the biopsy Storz® device without sample collection. In Experiment 2, pre-synchronised Charbray heifers (n = 13, 2 to 3 years old, live weight 439.8 ± 17.2) were biopsied in the horn ipsilateral to the CL at Day 4 (B4 = 6) or at Day 7 (B7 = 7) post-oestrus. In both experiments, blood samples were taken from jugular/coccygeal vein throughout the oestrus cycle on Days 3, 4, 6, 7, 9, 11, 13, 16 and 19 (Day 0 = synchronised oestrous) for progesterone (P4) analysis. Signs of heat were visually detected daily and confirmed by ovarian ultrasound. For the PGF2α metabolite 13,14-dihydro-15-keto-PGF2α (PGFM) assessment, blood samples were collected before and after uterine trauma at –24, –12, 0 h and +6, +18, +24 h (Hour 0 = UM or EB either at Day 4 or Day 7). Progesterone was assessed by liquid chromatography-mass spectrometry (LC-MS) and PGFM using an enzyme immunoassay DetectX® kit (Arbor Assays, Ann Arbor, MI, USA). The intra- and interassay coefficients of variations were 3.7% and 12.8%, respectively. A series of generalized additive mixed effect models (GAMM) were created to quantify P4 differences for UM and EB. The results showed that oestrous cycle length was not affected by UM or EB conducted at Day 4 or at Day 7, because P4 concentration remained above 1 ng mL−1 after uterine trauma during dioestrus. Also, UM and EB did not induce the release of luteolytic pulses of PGF2α. Although P4 concentration followed the normal cycling pattern throughout the oestrus cycle, the shape of the curve in EB4 and UM4 heifers showed higher concentrations of P4 compared with EB7 and UM7 heifers. In conclusion, UM and EB with Storz® device during metoestrus or early dioestrus had minimal or nil effects on CL activity. Whether lower P4 profiles affect embryo development after uterine trauma needs to be further elucidated.

Attempting to extract useful and reliable information about semen quality and its fertility potential remains a difficult exercise, partly because the sperm heterogeneity within samples often renders simple statistical analyses rather meaningless. In fact, a mean and standard deviation may reflect neither the very fast swimming activities of the most active cells nor the slow and sluggish activities of others. Herein we propose that the information value within semen samples can be maximised if current knowledge about sperm activation mechanisms is exploited before undertaking the measurements. We explain, using boar semen as an example, that estimating and defining relative sperm subpopulation sizes, after activation by bicarbonate, provides a means of quantifying sperm quality. Although such estimates may indeed be related to in vivo fertility, the general approach also suggests potential new avenues that could be exploited for the elaboration of novel in vitro tests for the characterisation of toxic environmental chemicals and, indeed, to reduce the number of animals used in such testing programs.

Saccharin (SAC) is a synthetic sweetener used in soft drinks and candies. Consumption of excess SAC leads to several health problems. Therefore, developing a simple method to detect its concentration in food stuffs and soft drinks is very important. The present study describes a simple colorimetric method for SAC determination using graphene oxide-silver nanoparticles (GO-AgNPs) composite solution. The yellow color GO-AgNPs was changed to reddish brown while adding 600 μM SAC and the emission of GO was “turn off” after the formation of AgNPs. However, the emission was “turn on” after the addition of SAC. This was ascribed to the strong interaction between sulfonamide groups of SAC with AgNPs, resulting the leaching of AgNPs from GO surface. The emission intensity increases linearly when SAC concentration increases from 500 nM to 1 mM with R2 = 0.9971 and the limit of detection was 0.7 mg L−1 (S/N = 3). The present GO-AgNPs composite was also used to selectively determine SAC containing 1400-fold higher concentration of interferents. Finally, the real sample analysis was carried out by determining SAC in candies and soft drinks by using GO-AgNPs.

In cattle, endometrial gene expression during the first 2 weeks after fertilization is different in cattle that conceive compared with those that fail to conceive. The potential selection of animals based on endometrial marker expression before the day of embryo transfer may improve the efficiency of bovine embryo transfer. This study aimed to evaluate the morphological and molecular changes induced by endometrial biopsy in cycling tropically adapted beef cattle. In both experiments, a single biopsy was performed on the horn ipsilateral to the corpus luteum using a circular cup biopsy forceps. The uterus was collected after slaughter, and sections were taken adjacent to the biopsy site. Five heifers were not biopsied and acted as controls. In Experiment 1, presynchronised heifers (n = 10) were biopsied on Day 7 post-oestrus (B7) and then resynchronised with cloprostenol (Estromil®, 250 μg IM) to induce oestrus (O), 2 to 3 days after injection. At Day 7 post-oestrus (D7), the reproductive tracts were collected, 10 days after biopsy (B7-O-D7). In Experiment 2, presynchronised heifers (n = 7) were biopsied on Day 4 post-oestrus (B4), and reproductive tracts were collected 3 days later (B4-D7). A quantitative scoring system was used to define histological appearance of the endometrium (1 to 25; above 16 indicates endometritis). The RNA expressions of interleukin-1-β (IL-1β) and transforming growth factor-β (TGF-β) were measured by quantitative real-time PCR (qRT-PCR). Macroscopic examination showed the uterus collected from B7-O-D7 heifers exhibited a focal, well demarcated, dark red discoloration of the endometrium (4/10) with no evidence of the biopsy site found in the remaining heifers (6/10). The biopsied uteri from B4-D7 heifers revealed no macroscopic evidence of the biopsied site (4/7), serosa congestion (2/7), and one case where a severe endometrial haemorrhage was evident (1/7). Histologically, the overall mean score was not indicative of endometritis. The RNA expression showed up-regulation of both IL-1β and TGF-β in both B7-O-D7 and B4-D7 heifers compared with non-biopsied heifers and significantly higher expression of IL-1β in B4-D7 than B7-O-D7 (P = 0.031, P < 0.05). Although in most cases there was little or no evidence of damage to the endometrium within 3 to 10 days after endometrial biopsy, there was still some evidence of up-regulation of pro-inflammatory and tissue repair genes. What effect the latter has on likelihood of conception is yet to be determined.

An endometrial biopsy allows for a comprehensive assessment of the uterine environment of a breeding female. Although routine in mares, devices used for endometrial biopsies are impracticable in heifers due to the size and structure of the cervix. This report describes the use of a human bronchoscopy biopsy device (Karl Storz® 10366L) for collection of endometrial biopsies in Bos indicus beef heifers. The Storz® device is smaller and thinner and enabled the collection of an endometrial biopsy in 86% of heifers (n = 44/51). The biopsied tissue was of good quality and suitable for transcriptomic assessment of the endometrium, with total RNA yield and RNA integrity number (RIN) averaging 1.3 μg (range 0.4-5.3 μg) and 7.4 (range 5.7-8.4), respectively.