Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
ANIMAL SCIENCES
Non Technical Summary
Producers in the Southern states, such as Missouri, have to deal with heat stressed livestock during the summer. Heat stress decreases the animals production which leads to a loss of income for the producer. National economic losses due to decreased reproduction of livestock resulting from heat stress average approximately $543 million, $40 million, and $51 million annually for dairy, beef, and swine, respectively. Missouri producers alone concede an estimated $17 million, $2 million, and $5 million in dairy, beef, and swine industries, respectively, to reproduction losses. A large proportion of the losses are due to infertility of heat-stressed males. Previous studies from our laboratory have identified epididymal tissue damage and reduced number of offspring from heat-stressed male mice (24h, 35C) compared to non heat-stressed controls. The overall aim of this experiment is to identify practical interventions which reduce the negative effects of heat stress on male
fertility. Specifically, we seek: 1) to identify the mechanism through which Artemisia afra maintains nearly normal fertility in heat-stressed males; and 2) to determine if similar effects are elicited by the more readily available Artemisia absinthium. Four groups of ten male mice will be used in the study. Males (n = 5 per heat-stress treatment) will be implanted with abdominal temperature transmitters to record core body temperature. Males will be offered a tea of the test plants as their drinking water for five days before a 24 hour period of moderate heat stress. Remaining males will be hemicastrated to collect testes tissue for measurement of activity of specific genes previously identified as affected by heat stress. Fertility of all males will then be tested in two mating trials, one with two females from days 3-11 following heat stress (this period is to confirm fertility of males independent of the effects of heat stress), a period when no loss in fertility is expected, and
subsequently with eight females per male on days 18-28 following heat stress, a period when fertility is expected to be impaired. Feed and water consumption, body temperature, gene expression and number of pregnancies and progeny will be compared among five treatment groups: a non heat-stressed negative control, a non treated heat-stressed positive control, and two groups provided teas of Artemisia afra, or Artemisia absinthium.
Animal Health Component
30%
Research Effort Categories
Basic
30%
Applied
30%
Developmental
40%
Goals / Objectives
1) National economic losses from heat stress causing decreased reproduction average $543 million, $40 million, and $51 million annually for dairy, beef, and swine, respectively. Missouri producers concede approximately $17 million, $2 million, and $5 million in dairy, beef, and swine production, respectively, due to reproduction losses. 2) We have previously shown that heat-stress induced male infertility is lowly heritable and have identified genes differentially expressed between males susceptible versus resistant to heat stress. 3) We have also shown that pretreatment with Artemisia afra prevents heat-stress induced infertility in mice. Identification of the mechanism of effect of Artemisia, as well as possible alternatives to Artemisia afra, will move the livestock industry closer to a preventative for heat-stress induced infertility. Our hypothesis is that medicinal herb Artemisia afra, traditionally used for alleviating heat stress symptoms in humans, will reduce
the negative effects of heat stress on male fertility. The overall aim of this experiment is to identify practical interventions which reduce the negative effects of heat stress on male fertility. The specific objectives of this study are: 1) to identify the mechanism through which Artemisia afra maintains nearly normal fertility in heat-stressed males; and 2) to determine if similar effects are elicited by the more readily available Artemisia absinthium.
Project Methods
Mature male mice will be implanted with Mini-Mitter temperature transmitters (n = 6 per treatment) to record core body temperature. Upon recovery from surgery, mice (n = 10 per treatment) will be placed in environmental chambers at control temperature (21+/-1C) for five days and provided tap water (control) or decoctions of Artemsia afra, or Artemisia absinthium. Males will be exposed to heat stress conditions (35+/-1C) for 24 h and then hemicastrated (d 1) to collect tissue for gene expression analyses. Two periods of mating tests will follow, the first from d 3-11 when no reduction in fertility is anticipated (two females/male) to confirm fertility, and the second from d 18-28 when a reduction in fertility in heat-stressed control males is expected (eight females/male). Pregnant females from the second mating period will be sacrificed at d 17 of gestation to count number of fetuses. Total RNA will be extracted from testis tissue (collected d 1) of males from each
treatment, plus from non heat-stressed controls, with trizol according to the recommendations of the manufacturer. RNA samples will be treated with DNAse I (Ambion, TX) to eliminate DNA contamination. Following concentration and purification of the samples, RNA quality will be assessed by electrophoresis and spectrophotometry. Analyses using real-time RT-PCR to confirm and quantify relative gene expression differences will be performed on calnexin, heat shock 90kDa protein 1 beta, t-complex protein 1, FK506-binding protein 6, proteosomal subunit 7, and isocitrate dehydrogenase 1. RNA will be reverse transcribed into cDNA using gene specific primers and the amplification kit from Invitrogen. The PE Biosystems ABI Prism 7700 Detection System (5' nuclease detection assay; TaqMan PCR Core Reagents Kit, PE Biosystems)18 will then be utilized to quantify mRNA expression. To avoid potential non-specific amplifications, internal fluorogenic probes will be used. Standard curves and the
fluorescent threshold will be established for every gene. Effects of treatment (no heat stress control, heat stress control, Artemisia afra, or Artemisia absinthium) on feed and water consumption, weight gain, number of females mated and number of fetuses will be analyzed using one-way analysis of variance in PROC GLM of SAS. Effects on body temperature will be will be analyzed using repeated measures analysis of variance in PROC MIXED of SAS. For variable for which treatment effects are significant, post-test contrasts of 1) heat-stress versus control, 2) heat stress control versus Artemisia treated, and 3) Artemisia afra versus Artemisia absinthium will be conducted.