Time and temperature stability of Tritrichomonas foetus in phosphate-buffered saline as evaluated by a reverse transcription real-time PCR assay and field analysis

Tritrichomonas foetus (TF) is a significant reproductive pathogen of cattle, and sample collection, handling, transport, and testing are significant hurdles to surveillance programs. Recent methods have been developed that allow for the direct detection of TF using a reverse transcription real-time PCR (direct RT-qPCR) approach. To evaluate these methods, a comparative analysis was conducted to assess the technical performance of this assay with a commercially available real-time PCR (qPCR) assay. In addition, the evaluation of two types of collection media (PBS and TF transport tube) was conducted that evaluated sample stability from 0 to 3 days when stored at 4°C or 25°C. Extended incubation times for PBS media were also evaluated (5, 7, and 14 days) at both refrigeration and frozen temperatures to evaluate the effect of extended transport time on samples. Limits of detection (LODs), dynamic range, and RNA stability were assessed using lab-cultured TF spiked into samples of normal bovine smegma collected in PBS or TF transport media, and performance was assessed on field samples collected in parallel. 100% agreement was found between direct RT-qPCR and qPCR at 10 parasites/extraction and a LOD of 1 parasite/extraction. Differences in detection were not observed in either collection media when incubated at either temperatures for up to 3 days of incubation. In addition, the extended incubation experiments indicate that samples containing 10 parasites/extraction can be detected at 4°C for 5 days with a mean Cq 26.34 (95% CI: 23.11–29.58) and detected at −20°C for 7 or 14 days, with a mean Cq 29.55 (95% CI: 27.73–31.37). A significant decrease in detectable RNA was observed in samples containing <10 parasites/extraction at −20°C for 14 days, which should be considered for long-term storage. In summary, direct RT-qPCR was found to be equivalent or superior to qPCR and PBS was not significantly different from TF transport media. The findings of the current study allows for more flexibility during sample collection and transport and ultimately enhancement of TF surveillance programs

COWS WITH ANDROGEN EXCESS IN FOLLICULAR FLUID HAVE ALTERED REPRODUCTIVE CYCLES, SEX HORMONE BINDING GLOBULIN, METABOLISM AND GONADOTROPIN SECRETION

Cow fertility is influenced by many factors. Two different studies were conducted with eleven different cows to better understand the mechanisms associated with infertility in cows with excess androgen in follicular fluid in their dominant follicle (High A4) compared to control counterparts (Low A4). The first study evaluated three different estrous cycles and determined that there was no difference in estrous cycle length. However, cows with High A4 ovulated without displaying estrus and/or displayed estrus without ovulating demonstrating that they had irregularities in their reproductive cycle. High A4 cows also had less circulating sex hormone binding globulin (SHBG), reduced circulating concentrations of LH and FSH, lower androstenedione (A4) and nonsterified fatty acids (NEFA) in plasma and altered rise and reduction in progesterone when compared to Low A4 cows. The objective of study 3 was to evaluate the response of these groups of cows to FSH and collect data at ovariectomy. High A4 cows had fewer granulosa cells per mL of follicular fluid and/or per follicles, different lipid composition in blood plasma during a non-stimulated cycle and after FSH stimulation in plasma and in follicular fluid when compared to Low A4 controls. In summary, these studies demonstrate that cows with androgen excess: (1) can ovulate without displaying estrus, and/or can display estrus but be anovulatory; (2) have different endocrine and metabolic profiles; (3) have impaired granulosa cell function potentially due to reduced number; and (4) have different lipid composition in blood plasma and follicular fluid. Thus, many of these alterations in endocrine and metabolic function may be contributing to reduced fertility in these High A4 cows. Advisor: Andrea S. Cup

VEGFA: Just one of multiple mechanisms for Sex-Specific Vascular Development within the testis?

Testis development from an indifferent gonad is a critical step in embryogenesis. A hallmark of testis differentiation is sex-specific vascularization which occurs as endothelial cells migrate from the adjacent mesonephros into the testis to surround Sertoli-germ cell aggregates and induce seminiferous cord formation. Many in vitro experiments have demonstrated that Vascular Endothelial Growth Factor A (VEGFA) is a critical regulator of this process. Both inhibitors to VEGFA signal transduction and excess VEGFA isoforms in testis organ cultures impaired vascular development and seminiferous cord formation. However, in vivo models using mice which selectively eliminated all VEGFA isoforms: in Sertoli and germ cells (pDmrt1-Cre;Vegfa−/−); Sertoli and Leydig cells (Amhr2-Cre;Vegfa−/−) or Sertoli cells (Amh-Cre;Vegfa−/− and Sry-Cre;Vegfa−/−) displayed testes with observably normal cords and vasculature at postnatal day 0 and onwards. Embryonic testis development may be delayed in these mice; however, the postnatal data indicate that VEGFA isoforms secreted from Sertoli, Leydig or germ cells are not required for testis morphogenesis within the mouse. A Vegfa signal transduction array was employed on postnatal testes from Sry-Cre;Vegfa−/− versus controls. Ptgs1 (Cox1) was the only upregulated gene (5-fold). COX1 stimulates angiogenesis and upregulates, VEGFA, Prostaglandin E2 (PGE2) and PGD2. Thus, other gene pathways may compensate for VEGFA loss, similar to multiple independent mechanisms to maintain SOX9 expression. Multiple independent mechanism that induce vascular development in the testis may contribute to and safeguard the sex-specific vasculature development responsible for inducing seminiferous cord formation, thus, ensuring appropriate testis morphogenesis in the male

Time and temperature stability of \u3ci\u3eTritrichomonas foetus\u3c/i\u3e in phosphate-buffered saline as evaluated by a reverse transcription real-time PCR assay and field analysis

Tritrichomonas foetus (TF) is a significant reproductive pathogen of cattle, and sample collection, handling, transport, and testing are significant hurdles to surveillance programs. Recent methods have been developed that allow for the direct detection of TF using a reverse transcription real-time PCR (direct RT-qPCR) approach. To evaluate these methods, a comparative analysis was conducted to assess the technical performance of this assay with a commercially available real- time PCR (qPCR) assay. In addition, the evaluation of two types of collection media (PBS and TF transport tube) was conducted that evaluated sample stability from 0 to 3 days when stored at 4◦C or 25◦C. Extended incubation times for PBS media were also evaluated (5, 7, and 14 days) at both refrigeration and frozen temperatures to evaluate the effect of extended transport time on samples. Limits of detection (LODs), dynamic range, and RNA stability were assessed using lab-cultured TF spiked into samples of normal bovine smegma collected in PBS or TF transport media, and performance was assessed on field samples collected in parallel. 100% agreement was found between direct RT-qPCR and qPCR at 10 parasites/extraction and a LOD of 1 parasite/extraction. Differences in detection were not observed in either collection media when incubated at either temperatures for up to 3 days of incubation. In addition, the extended incubation experiments indicate that samples containing 10 parasites/extraction can be detected at 4◦C for 5 days with amean Cq 26.34 (95% CI: 23.11–29.58) and detected at −20◦C for 7 or 14 days, with a mean Cq 29.55 (95% CI: 27.73–31.37). A significant decrease in detectable RNA was observed in samples containing \u3c10 parasites/extraction at −20◦C for 14 days, which should be considered for long- term storage. In summary, direct RT-qPCR was found to be equivalent or superior to qPCR and PBS was not significantly different from TF transport media. The findings of the current study allows for more flexibility during sample collection and transport and ultimately enhancement of TF surveillance programs

Cows with Excess Androgen are Anovulatory and Have Differing Patterns of Progesterone Secretion

Within the physiology herd, a group of cows that have excess androgen (androstenedione, A4) in the dominant follicle and a 17% reduction in calving rate have being identified. Thus, our objective was to determine follicular dynamics (follicle growth) and progesterone (P4) concentrations in High A4 cows to determine if they were anovulatory. High A4 cows had more persistent dominant follicles and either did not display estrus and ovulated at an inappropriate time or did not ovulate compared with Low A4 cows (Controls). Furthermore, P4 concentrations had reduced peak values and were maintained longer in High vs Low A4 cows which may contribute to their failure to ovulate

The Effects of Androgens on Bovine and Human Granulosa Cells

A subpopulation of cows in the Physiology herd has been identified as subfertile due to sporadic or chronic anovulation. This decrease in fertility could be lost profits for farmers and raises questions about ovulation disorders in women. Granulosa cells, a type of cell that is essential to ovarian follicle development, was investigated to determine if high concentrations of a hormone, androstenedione, could impact the follicular environment enough to cause anovulation disorders. Previous studies suggested that excess androgen may decrease the number of functioning granulosa cells by preventing them from proliferating within the follicle. Fewer granulosa cells mean fewer cells that are available to convert androstenedione to estrogen; and estrogen is required for the development of the follicle and ovulation. This research experience determined that when primary granulosa cells are subjected to high concentrations of androstenedione the rate of proliferation decreases based on the reduced proliferation promoting genes within the cells. These tests were done on primary granulosa cells from bovine as well as an immortalized granulosa cell line from humans

Granulosa Cell Exposure to Excess Androgens Inhibits Their Ability to Proliferate in the Cow Which May Cause or Perpetuate Androgen Excess

Within the UNL physiology herd, a group of cows have been identified with excess androgen (androstenedione, A4) in their dominant follicle (30 fold higher than controls) and a 17% reduction in calving rate, suggesting subfertility. Th e objective was to identify altered granulosa cell gene expression that could be preventing these cells from converting excess androgen into estrogen. Microarray analysis suggests these granulosa cells experience inhibited proliferation resulting in a reduced total population of cells. Improved understanding of the causes of this phenotype may provide beef producers with tools to identify potentially subfertile cattle and improve reproductive efficiency

Maternal age influences the number of primordial follicles in the ovaries of yearling Angus heifers

The number of antral follicles detectable by ultrasonography in heifers is influenced by age of the dam, because daughters of primiparous cows have fewer antral follicles than daughters of mature cows. We, therefore, hypothesized that heifers with primiparous dams would have fewer primordial follicles in their ovaries than heifers born to mature (4+ y) cows. Angus heifers (n=464) were submitted for ultrasonographic evaluation of antral follicle number at 325, 355, and 385 d of age. Ovaries were collected from a random subset of heifers (n=79) and processed for histological evaluation to determine number of primordial follicles. A greater percentage of heifers with primiparous dams had a corpus luteum at first ultrasonographic examination; however, a greater percentage of heifers with multiparous dams had ovulated by the start of breeding (P \u3c 0.01). Heifers with primiparous dams had fewer antral follicles detectable by ultrasonography (P \u3c 0.01). Heifers with primparous dams had fewer surface antral follicles on their ovaries (P \u3c 0.01), and the number of primordial follicles per histological section was less for heifers with primiparous dams (P= 0.02). These data indicate that the lesser number of antral follicles detectable by ultrasonography in heifers with primparous dams is due to less ovarian follicle reserves. Selecting replacement heifers from mature dams may result in daughters with greater fertility and reproductive longevity; however, further research is necessary to determine if interactions between size of the ovarian follicle reserve and age at puberty influence fertility and reproductive longevity in replacement heifers