Rhinoceros horn mineral and metal concentrations vary by sample location, depth, and color.

Poaching is again driving rhinos to the brink of extinction due to the demand for rhino horn products consumed for cultural, medicinal, and social purposes. Paradoxically, the same horn for which rhinos are killed may contain valuable clues about the species' health. Analyses of horn composition could reveal such useful bioindicators while elucidating what people actually ingest when they consume horn derivatives. Our goals were to quantify minerals (including metals) in rhino horn and investigate sampling factors potentially impacting results. Horns (n = 22) obtained during necropsies of white (n = 3) and black (n = 13) zoo rhinos were sampled in several locations yielding 182 specimens for analysis. Initial data exposed environmental (soil) contamination in the horn's exterior layer, but also confirmed that deep (≥ 1 cm), contaminant-free samples contained measurable concentrations of numerous minerals (n = 18). Of the factors examined in deep samples, color-associated mineral differences were the most profound with dark samples higher in zinc, copper, lead, and barium (p < 0.05). Our data demonstrate that rhino horns contain both essential and potentially toxic minerals that could be relevant to rhino health status, but low concentrations make their human health benefits or risks unlikely following consumption.

Heat-induced increases in body temperature in lactating dairy cows: impact on the cumulus and granulosa cell transcriptome of the periovulatory follicle.

Cows acutely heat stressed after a pharmacologically induced luteinizing hormone (LH) surge had periovulatory changes in the follicular fluid proteome that may potentiate ovulation and impact oocyte developmental competence. Because the cellular origins of differentially abundant proteins were not known, we have examined the cumulus and granulosa cell transcriptomes from the periovulatory follicle in cows exhibiting varying levels of hyperthermia when occurring after the LH surge. After pharmacological induction of a dominant follicle, lactating dairy cows were administered gonadotropin releasing hormone (GnRH) and maintained in thermoneutral conditions (~67 temperature-humidity index [THI]) or heat stress conditions where THI was steadily increased for ~12 h (71 to 86 THI) and was sufficient to steadily elevate rectal temperatures. Cumulus-oocyte complexes and mural granulosa cells were recovered by transvaginal aspiration of dominant follicle content ~16 h after GnRH. Rectal temperature was used as a continuous, independent variable to identify differentially expressed genes (DEGs) increased or decreased per each 1 °C change in temperature. Cumulus (n = 9 samples) and granulosa (n = 8 samples) cells differentially expressed (false discovery rate [FDR] < 0.05) 25 and 87 genes, respectively. The majority of DEGs were upregulated by hyperthermia. Steady increases in THI are more like the "turning of a dial" than the "flipping of a switch." The moderate but impactful increases in rectal temperature induced modest fold changes in gene expression (<2-fold per 1 °C change in rectal temperature). Identification of cumulus DEGs involved in cell junctions, plasma membrane rafts, and cell-cycle regulation are consistent with marked changes in the interconnectedness and function of cumulus after the LH surge. Depending on the extent to which impacts may be occurring at the junctional level, cumulus changes may have indirect but impactful consequences on the oocyte as it undergoes meiotic maturation. Two granulosa cell DEGs have been reported by others to promote ovulation. Based on what is known, several other DEGs are suggestive of impacts on collagen formation or angiogenesis. Collectively these and other findings provide important insight regarding the extent to which the transcriptomes of the components of the periovulatory follicle (cumulus and mural granulosa cells) are affected by varying degrees of hyperthermia.

Approximately 70% of the world's cattle population reside under ambient conditions experiencing some level of heat stress. Heat-stressed cows chronically exposed to elevated ambient temperatures have difficulty getting pregnant. Although the underlying basis for poor fertility during bouts of chronic heat stress remains unclear and is likely because of many different factors, when ambient conditions are sufficient to increase cow body temperature, different ovulatory follicle components are affected (i.e., mural granulosa cells that line the ovulatory follicle, the intrafollicular fluid and or the cumulus-oocyte complex while it matures in preparation for fertilization while resident within). To test this hypothesis, we have examined the cumulus and granulosa cell transcriptomes from the periovulatory follicle in cows. Using steady increases in THI to induce varying levels of elevated body temperature after the luteinizing hormone surge we discovered certain genes in the cumulus cells that may have indirect but impactful consequences on the oocyte as it undergoes meiotic maturation. We also noted changes in gene expression in granulosa cells that may impact ovulation and corpus luteum formation.

Impact of an acute heat shock during in vitro maturation on interleukin 6 and its associated receptor component transcripts in bovine cumulus-oocyte complexes.

An acute heat stress event after the LH surge increased interleukin 6 (IL6) levels in the follicular fluid of the ovulatory follicle in hyperthermic cows. To examine direct consequences of a physiologically-relevant elevated temperature (41.0°C) on the cumulus-oocyte complex (COC), IL6 transcript abundance and related receptor components were evaluated throughout in vitro maturation. Heat-induced increases in IL6 were first noted at 4 hours of in vitro maturation (hIVM); peak levels occurred at 4.67 versus 6.44 hIVM for 41.0 and 38.5°C COCs, respectively (SEM = 0.23; P < 0.001). Peak IL6ST levels occurred at 6.95 versus 8.29 hIVM for 41.0 and 38.5°C, respectively (SEM = 0.23; P < 0.01). Transcript for LIF differed over time (P < 0.0001) but was not affected by 41.0°C exposure. Blastocyst development after performing IVF was not affected by 41.0°C exposure for 4 or 6 h. When limiting analysis to when IL6 was temporally produced, progesterone levels were only impacted by time and temperature (no interaction). Heat-induced shift in the temporal production of IL6 and IL6ST along with its impact on progesterone likely cooperate in heat-induced hastening of meiotic progression described by others.

The effects of protein level on cytokines and chemokines in the uterine environment of beef heifers during development.

The development of replacement heifers is crucial for breeding success and herd efficiency. Nutritional management can affect not only reproductive development but also the inflammatory status of the uterine environment, which may impact reproductive functions such as pregnancy establishment and development. The study herein evaluated the concentration of cytokines and chemokines in the uterus of heifers supplemented with different levels of protein. Angus heifers (n = 60) were blocked by body weight (BW) and randomly assigned to 1 of 3 treatments based on protein supplementation level: control of 10% crude protein (CON), 20% crude protein (P20), or 40% crude protein (P40). BW, body condition score, and blood samples were taken every 2 wk for 140 d to monitor development. Uterine flushes were performed monthly and concentrations of cytokines (IL-1α, IL-1ß, TNF-α, IFN-γ, IL-10, VEGF-α, IL-17A, and IL-36RA) and chemokines (IL-8, MCP-1, MIP-1α, and MIP-1ß) were quantified via ELISA multiplex. To test if there were mean differences in cytokines between the treatment groups or over time, PROC GLIMMIX (SAS v 9.4) was utilized. Concentrations of all cytokines and chemokines, except IL-1α, changed throughout heifer development (P < 0.05). Heifers in the P40 treatment group displayed reduced concentrations of MCP-1 (P = 0.007) and tended to have decreased concentrations of IFN-γ (P = 0.06). Cytokine IL-36RA tended (P = 0.06) to be affected by protein level, with the lowest concentrations observed in CON heifers. Most cytokines and chemokines increased following the initial month of supplementation (P < 0.05). The increase in concentrations after 1 mo may indicate an adaptive response in the uterus to diet change. Cytokines and chemokines fluctuated due to physiological changes occurring during development. Further research is needed to determine the influence of nutrition on uterine inflammation and long-term impacts on reproductive function.

Heat-induced hyperthermia impacts the follicular fluid proteome of the periovulatory follicle in lactating dairy cows.

We hypothesized that heat-induced perturbations in cumulus cells surrounding the maturing oocyte may extend to the mural granulosa of the periovulatory follicle in the heat-stressed cow to subsequently the follicular fluid proteome. Lactating Holsteins were pharmacologically stimulated to have a dominant follicle that was capable of responding to a gonadotropin releasing hormone-induced luteinizing hormone surge. Following gonadotropin releasing hormone administration, cows were maintained at ~67 temperature humidity index (THI; thermoneutral conditions) or exposed to conditions simulating an acute heat stress event (71 to 86 THI; heat stress for ~12 h). Dominant follicle collection was conducted in the periovulatory period ~16 h after gonadotropin releasing hormone. Follicular fluid proteome from thermoneutral (n = 5) and hyperthermic (n = 5) cows was evaluated by quantitative tandem mass spectrometry (nano LC-MS/MS). We identified 35 differentially-abundant proteins. Functional annotation revealed numerous immune-related proteins. Subsequent efforts revealed an increase in levels of the proinflammatory mediator bradykinin in follicular fluid (P = 0.0456) but not in serum (P = 0.9319) of hyperthermic cows. Intrafollicular increases in transferrin (negative acute phase protein) in hyperthermic cows (P = 0.0181) coincided with a tendency for levels to be increased in the circulation (P = 0.0683). Nine out of 15 cytokines evaluated were detected in follicular fluid. Heat stress increased intrafollicular interleukin 6 levels (P = 0.0160). Whether hyperthermia-induced changes in the heat-stressed cow's follicular fluid milieu reflect changes in mural granulosa, cumulus, other cell types secretions, and/or transudative changes from circulation remains unclear. Regardless of origin, heat stress/hyperthermia related changes in the follicular fluid milieu may have an impact on components important for ovulation and competence of the cumulus-oocyte complex contained within the periovulatory follicle.

An in vivo model to assess the thermoregulatory response of lactating Holsteins to an acute heat stress event occurring after a pharmacologically-induced LH surge.

Hyperthermia occurring 10-12 h after LH surge reduces quality of maturing oocyte, thereby reducing fertility. Objective was to examine consequences of an acute heat stress and the influence of certain hormones on the thermoregulatory responses of lactating cows during this critical period. Between the months of February through May, cows were transported to a facility and maintained at a temperature-humidity index (THI) of 65.9 ±â€¯0.2 (thermoneutral) or exposed to changes in THI to simulate what may occur during an acute heat stress event (71-86 THI; heat stress); cows were rapidly cooled thereafter. Mixed model regressions with repeated measures were used to test respiration rates (RR) and rectal temperature (RT). Within 40 and 110 min of increasing THI, RR increased in a quadratic fashion (P < 0.001); RT increased by 0.04 ±â€¯0.1 °C (P < 0.001) per unit THI. Changes in RR lagged THI and preceded rises in RT. Average THI 3-days before treatment (prior THI) influenced RR (P = 0.050) and RT (P < 0.001) changes. Increased RR was more noticeable in heat-stressed cows when prior THI was in the 40 s. Rectal temperature of heat-stressed cows was 0.8 ±â€¯0.02 °C lower when prior THI was in the 40 s versus low 60 s. Levels of progesterone and luteinizing hormone before treatment were predictive of thermoregulatory response in heat-stressed cows. Rapid cooling decreased RR by 0.6 ±â€¯0.1 bpm (P < 0.001) and RT by 0.02 ±â€¯0.002 °C per min (P < 0.002). Speed and magnitude of thermoregulatory changes to an acute heat stress and after sudden cooling emphasizes importance of strategic cooling before ovulation. Efforts to do so when prior THI approaches levels expected to induce mild stress are especially important. Respiration rate is a useful indicator of the degree of hyperthermia a lactating cow is experiencing.

Heat stress impairs gap junction communication and cumulus function of bovine oocytes.

The intimate association of cumulus cells with one another and with the oocyte is important for regulating oocyte meiotic arrest and resumption. The objective of this study was to determine the effects of heat stress on cumulus cell communication and functions that may be related to accelerated oocyte meiosis during early maturation. Bovine cumulus-oocyte complexes underwent in vitro maturation for up to 6 h at thermoneutral control (38.5°C) or elevated (40.0, 41.0 or 42.0°C) temperatures. Gap junction communication between the cumulus cells and the oocyte was assessed using the fluorescent dye calcein after 4 h of in vitro maturation. Dye transfer was reduced in cumulus-oocyte complexes matured at 41.0°C or 42.0°C; transfer at 40.0°C was similar to control (P < 0.0001). Subsequent staining of oocytes with Hoechst revealed that oocytes matured at 41.0 or 42.0°C contained chromatin at more advanced stages of condensation. Maturation of cumulus-oocyte complexes at elevated temperatures reduced levels of active 5' adenosine monophosphate activated kinase (P = 0.03). Heat stress exposure had no effect on active extracellular-regulated kinase 1/2 in oocytes (P = 0.67), associated cumulus cells (P = 0.60) or intact cumulus-oocyte complexes (P = 0.44). Heat-induced increases in progesterone production by cumulus-oocyte complexes were detected during the first 6 h of maturation (P = 0.001). Heat-induced alterations in gap junction communication and other cumulus-cell functions likely cooperate to accelerate bovine oocyte meiotic progression.

Mitochondrial-related consequences of heat stress exposure during bovine oocyte maturation persist in early embryo development.

Hyperthermia during estrus has direct consequences on the maturing oocyte that carries over to the resultant embryo to compromise its ability to continue in development. Because early embryonic development is reliant upon maternal transcripts and other ooplasmic components, we examined impact of heat stress on bovine oocyte transcripts using microarray. Oocytes were matured at 38.5ºC for 24 h or 41.0ºC for the first 12 h of in vitro maturation; 38.5ºC thereafter. Transcriptome profile was performed on total (adenylated + deadenylated) RNA and polyadenylated mRNA populations. Heat stress exposure altered the abundance of several transcripts important for mitochondrial function. The extent to which transcript differences are coincident with functional changes was evaluated by examining reactive oxygen species, ATP content, and glutathione levels. Mitochondrial reactive oxygen species levels were increased by 6 h exposure to 41.0ºC while cytoplasmic levels were reduced compared to controls (P < 0.0001). Exposure to 41.0ºC for 12 h increased total and reduced glutathione levels in oocytes at 12 h but reduced them by 24 h (time × temperature P < 0.001). ATP content was higher in heat-stressed oocytes at 24 h (P < 0.0001). Heat-induced increases in ATP content of matured oocytes persisted in early cleavage-stage embryos (8- to 16-cell embryos; P < 0.05) but were no longer apparent in blastocysts (P > 0.05). Collectively, results indicate that direct exposure of maturing oocytes to heat stress may alter oocyte mitochondrial processes/function, which is inherited by the early embryo after fertilization.

Developmental consequences of supplementing with matrix metallopeptidase-9 during in vitro maturation of heat-stressed bovine oocytes.

Because latent form of matrix metallopeptidase-9 (proMMP9) levels are positively related to blastocyst development, it was hypothesized that addition during maturation may improve development of heat-stressed oocytes. To test hypothesis, 0, 30 or 300 ng/ml human proMMP9 (hMMP9) was added at 18 h of in vitro maturation (hIVM) to cumulus-oocyte complexes matured at 38.5 or 41.0ºC (first 12 h only). Heat stress decreased 24 hIVM proMMP9 levels only in 0 and 30 ng/ml groups and increased progesterone in 0 and 300 ng/ml hMMP9 groups. Heat stress decreased cleavage and blastocyst development. Independent of maturation temperature, hMMP9 at 18 hIVM decreased blastocyst development. In a second study, cumulus-oocyte complexes were matured for 24 h at 38.5 or 41.0ºC (HS first 12 h only) with 0 or 300 ng/ml hMMP9 added at 12 hIVM. Without hMMP9, heat stress decreased 24 hIVM proMMP9 levels and increased progesterone production. Addition of 300 ng/ml of hMMP9 produced equivalent levels of proMMP9 at 24 hIVM (271 vs. 279 ± 77 for 38.5ºC and 41.0ºC treated oocytes, respectively). Heat stress did not affect ability of oocytes to cleave but reduced blastocyst development. Independent of temperature, hMMP9 decreased cleavage and blastocyst development. In summary, hMMP9 supplementation during IVM did not improve development of heat-stressed oocytes even when it was added for the entire maturation period. At doses tested, hMMP9 appeared detrimental to development when supplemented during the last 12 or 6 h of oocyte maturation.

Impact of heat stress on germinal vesicle breakdown and lipolytic changes during in vitro maturation of bovine oocytes.

Two studies were conducted with the overarching goal of determining the extent to which lipolytic changes relate to germinal vesicle breakdown (GVBD) in bovine oocytes matured under thermoneutral or hyperthermic conditions. To this end, cumulus-oocyte complexes underwent in vitro maturation for 0, 2, 4, 6 or 24 h at 38.5 (first study) or 38.5 and 41.0 C (second study; heat stress applied up through first 12 h only, then shifted to 38.5 C). Independent of maturation temperature, triglyceride and phospholipid content decreased markedly by 2 h of in vitro maturation (hIVM; P < 0.0005). Content was lowest at 24 hIVM with no detectable impact of heat stress when exposure occurred during first 12 hIVM. Germinal vesicle breakdown occurred earlier in oocytes experiencing heat stress with effects observed as soon as 4 hIVM (P < 0.0001). Germinal vesicle breakdown was associated with lipolytic changes (R(2) = 0.2123 and P = 0.0030 for triglyceride content; R(2) = 0.2243 and P = 0.0026 for phospholipid content). ATP content at 24 hIVM was higher in oocytes experiencing heat stress (P = 0.0082). In summary, GVBD occurs sooner in heat-stressed oocytes. Although marked decreases in triglyceride and phospholipid content were noted as early as 2 hIVM and preceded GVBD, lipolytic changes such as these are not likely serving as an initial driver of GVBD in heat-stressed oocytes because changes occurred similarly in oocytes matured at thermoneutral conditions.

Impact of heat stress exposure during meiotic maturation on oocyte, surrounding cumulus cell, and embryo RNA populations.

To determine if reductions in developmental competence related to heat stress exposure were correlated with perturbations in certain RNA populations, poly(A) RNA, total RNA, RNA size distribution, and the abundance of transcripts (cyclin B1, GDF9, BMP15, poly(A) polymerase, HSP70, 18S & 28S rRNA) were examined in oocytes matured at 38.5 or 41 C. Performing in vitro fertilization resulted in embryos for examining RNA. Relative to germinal vesicle-stage oocytes, total amount of poly(A) RNA decreased similarly in oocytes matured at 38.5 or 41 C. Total RNA did not change during meiotic maturation or up through the 4 to 8-cell stage of embryonic development. Blastocyst-stage embryos had more total RNA; those originating from heat-stressed oocytes had more than those from nonheat-stressed oocytes. Oocytes and 4 to 8-cell embryos had similar RIN values and ratios for rRNA, 18S/fast region, and 18S/inter region. Values obtained for blastocyst-stage embryos were similar to those obtained for cumulus cell RNA, which did not change during maturation. Culture at 41 C for the first 12 h of meiotic maturation had no impact on RNA size distribution or transcripts examined from oocytes, surrounding cumulus or resultant 4 to 8-cell embryos. Interestingly, however, RNA from blastocysts originating from heat-stressed oocytes had lower 18S/fast region and 18S/inter region ratios compared to other developmental stages and cumulus cells. Although biological significance of these RNA changes is unclear, differences at the molecular level in embryos from heat-stressed oocytes emphasize the importance of minimizing stress exposure during meiotic maturation, if the intent is to obtain developmentally-competent embryos.

General features of certain RNA populations from gametes and cumulus cells.

Results described herein provide insight regarding certain features of gamete RNA and how they compare to cumulus cell RNA. In particular, 28S/18S rRNA ratio and size distribution of RNA molecules differed in total RNA from oocytes versus surrounding cumulus cells. Specifically, oocyte total RNA had a lower rRNA ratio and an increased abundance of smaller RNA sizes compared to RNA from surrounding cumulus. Extensive efforts demonstrated that observed differences were repeatable whether oocyte maturation occurred in vitro or in vivo, and were similar between the nuclear stages examined. Features of oocyte RNA were conserved across six mammalian species, yet differed from surrounding cumulus. Profiles of sperm RNA were also examined but had no discernible ribosomal RNA peaks and were conserved across four mammalian species. Because the oocyte and spermatozoon are highly specialized cells representing unique molecular entities required for proper embryo development, dissimilarities described herein likely represent real gamete versus cumulus RNA differences.

Insight into the neuroendocrine site and cellular mechanism by which cortisol suppresses pituitary responsiveness to gonadotropin-releasing hormone.

Stress-like elevations in plasma glucocorticoids rapidly inhibit pulsatile LH secretion in ovariectomized sheep by reducing pituitary responsiveness to GnRH. This effect can be blocked by a nonspecific antagonist of the type II glucocorticoid receptor (GR) RU486. A series of experiments was conducted to strengthen the evidence for a mediatory role of the type II GR and to investigate the neuroendocrine site and cellular mechanism underlying this inhibitory effect of cortisol. First, we demonstrated that a specific agonist of the type II GR, dexamethasone, mimics the suppressive action of cortisol on pituitary responsiveness to GnRH pulses in ovariectomized ewes. This effect, which became evident within 30 min, documents mediation via the type II GR. We next determined that exposure of cultured ovine pituitary cells to cortisol reduced the LH response to pulse-like delivery of GnRH by 50% within 30 min, indicating a pituitary site of action. Finally, we tested the hypothesis that suppression of pituitary responsiveness to GnRH in ovariectomized ewes is due to reduced tissue concentrations of GnRH receptor. Although cortisol blunted the amplitude of GnRH-induced LH pulses within 1-2 h, the amount of GnRH receptor mRNA or protein was not affected over this time frame. Collectively, these observations provide evidence that cortisol acts via the type II GR within the pituitary gland to elicit a rapid decrease in responsiveness to GnRH, independent of changes in expression of the GnRH receptor.