environment hormones in hula valley

This was a chapter for a book on the Hula that was never published.

Source and transport of natural and synthetic steroid hormones in Hula Valley and their  effects on fish in aquiculture.

L. Shore*, Ph.D, Kimron Veterinary Institute, Bet Dagan, POB 12, Israel

 Key words:  testosterone, ethinylestradiol, estrogen, Cyprinus carpio 

Abstract

Natural and synthetic steroid hormones are constantly being excreted into the environment.  In the Hula Valley, five steroidal compounds were identified in the surface waters – testosterone, estrogen (estradiol-17b and estrone), androstenedione, ethinyl estradiol and estriol.  The sources of the hormones were shown to be cattle pasture and treated sewage effluent and the sources could be identified based on hormone profile.  However, of the five steroids tested, only testosterone could permeate the soil to reach the groundwater.  Fish with male gonadal atrophy but normal female gonads were found in a reservoir used for aquaculture. The gonadal atrophy was associated with the presence of hormones, specifically the synthetic hormone, ethinyl estradiol, which is used in contraceptive pills.    When in the subsequent year, the same fish were grown in the absence of the hormone, the males were found to have normal gonads.  

Introduction

In the past decade, many concerns have been expressed on compounds that mimic hormones and can disrupt reproduction in animals including humans (see Lintelmann et al., 2003 for review).  Of particular concern are synthetic hormones that are specifically design to act on biological systems.  All of these compounds act against a background of naturally produced hormones such as testosterone and estradiol that are constantly release into the environment (see Shore and Shemesh, 2003 for review).  Since our initial observation of the presence of significant amounts of testosterone and estrogen in Lake Kinneret in 1993 (Shore et al. 1993), we have surveyed the Jordan Valley including the Upper Jordan River Catchment and the Southern Jordan River for the presence of steroidal hormones (Bar-El Cohen et al., 2005; Shore et al.. 2004).   The present paper will described the results of these surveys in the Hula Valley as to the determination of sources, the pathways of transport and the effects on fish. 

The primary steroid hormones excreted into the environment are estrone, estradiol-17β, testosterone and androstenedione (Fig. 1). The natural steroids of major concern are estrone and estradiol since they exert their physiological effects at lower concentrations than other steroids and can be found in the environment in concentrations above their Lowest Observed Effect Level (LOEL) for fish (increased vitellogenin) and plants (increased growth) (10 ng/l) (Christiansen, 2002; Shore et al., 1992,1995b). In rivers and soil, estradiol is converted abiotically to estrone, so for environmental studies estradiol and estrone can be considered together as “estrogens” (Colucci et al., 2001; Jürgens et al., 2002). Effluent from human sources can also contain estriol, a weak estrogen excreted in the urine of pregnant women, and synthetic estrogens such as mestranol and ethinylestradiol (Fig. 1) (Wenzel et al., 1998). These synthetic compounds are of particular concern as they have LOEL’s in the order of 1 ng/l (Christiansen et al., 2003).

To study the occurrence, origin and effects of the natural and synthetic steroids, a general survey of the Hula Valley catchment area was performed as well as more in depth studies, at two sites to (1) determine nature of export of hormones from cattle pasture and (2) determine the effect of the hormones on fish raised in aquaculture.

Materials and methods:

Extractions and assays

Manures: Extraction and determination of testosterone, and estrogen in cattle manure were done by established procedures (Shore and Shemesh, 1993; Shore et al., 1993). Androstenedione was determined using a commercial ELISA (DRG GmbH, Marburg, Germany). 

Water: All water samples were collected into acid-washed polyethylene 1 L bottles, and immediately put in a cooler before transporting to the laboratory for further processing.  Extraction and determination of testosterone, estrogen, estriol and ethinylestradiol were done as previously described (Shore et al, 2004).

Soil:  Five gram samples were extracted twice with 15 ml of ethyl acetate, the supernatants combined, evaporated and redisolved in 0.5 ml of methanol.   100 µl aliquots were evaporated to dryness and redisolved in the testing media for analysis.

Statistics: All data are in means ± SD. Student t test used for comparing two groups.  

Figure 1.  Structures of natural and synthetic environmental steroids

Site description

The Hula Valley catchment area is divided by the Jordan River into a Western and Eastern sub-catchment areas.  The Eastern portion (Golan Heights, eastern part of the Hula valley) is characterized by the confluence of the Dan, Hermon and Sneir tributaries at the Joseph Bridge to form the Jordan River, which receives the runoff from the Golan Heights until it leaves the Hula Valley at a second confluence, the Huri Bridge.   At the Joseph Bridge, about 10% of the Jordan River is diverted to the Western Canal which receives runoff from the western or Naphtali Heights.  The Western Canal rejoins the Jordan shortly before its exit from the Hula Valley. Precipitation in the Hula Valley catchment area occurs only in the rainy season from Oct. to May. The area consists of small farms, cattle pasture, fishponds with some urban development.  The water table is shallow being less than 2 meters from the surface. The fifteen sites sampled after each rain event (11 to 12 times a hydrological year) on the eastern side of the catchment are shown in Figure 2.  The sites samples on the western side were in two separate subcatchments (points P, KD on  Fig. 3)  which were sampled on two occasions (Jan. 24, Feb 2, 2005) following rain events. For the aquiculture observations, a reservoir containing 400 tons of three species of fish (grey mullet, carp and tilapia) was sampled to determine the effect on aquaculture of exposure to sewage effluent (site 404/2, fig. 3).  The density of the fish was approximately 1 fish/l.  The reservoir held 5.6 mcu³ over area of 100 acres and a depth of 15 m. The reservoir received water from three sources: sewage effluent from an urban area of about 8000, excess water flow from Lake Hula  and Jordan River water from the Western canal. The limonological properties of the reservoir have been described in detail (Gafny et al., 1999). Samples were taken on 16.02.05 before fish were introduced and 04.05.05, 04.08.05 and 19.11.05 representing 1, 5 and 8 months after the introduction. Samples were taken at depths of 1, 3 and 6 m except on the final sampling before harvesting when the depth was only 0.8 m. Samples were taken from a control reservoir without fish on the same dates.  In Nov. 2005, the remaining males were transferred to another pond containing the same water without any sewage contaminant. Gonadal somatic index (GSI) for the carp was evaluated according to Degani et al. (1996, 1998).

For the cattle pasture study, a fenced field in which cattle were not present most of the year with a pool receiving runoff from the field was studied after the introduction of the cattle for three months. The grazing intensity was 0.63 cows/ha. For edge of field drainage, one liter samples in polyethylene bottles were obtained from the receiving pool after each of three rain events. Samples were acidified and transported to the laboratory.  Single samples of soil were taken after 1 mo., 2 mo and 3 mo. after introduction of cattle at a depth of 10, 20 and 30 cm.

Figure 2.  Sites in the Hula Valley draining the Eastern side of the catchment sampled in the hydrological years 2001/2 and 2002/3. The sites are listed in Table 1 found below.

Figure 3. Two subcatchments (Di, Kd) draining cattle pasture in the western portion of Hula Valley catchment studied in hydrological year 2003/2004.  The defined cattle pasture studied for soil and manure samples was located at N2 (Site #1). The reservoir used for aquaculture was at site 404/2.

Results

Eastern part of the Hula Valley

The transport of testosterone, estrogen, ethinylestradiol and estriol was measured at fifteen sites in the eastern side of Hula Valley catchment area after major rain events (10 -12 per hydrological year).  It was found in the rain season of 2001/2002, that following a rain sequence of 131 mm/wk, there was an initial large increase in the concentration of testosterone (maximum 6 ng/l) accompanied by high estrogen (maximum 6 ng/l), which then gradually declined to non-detectable levels (<0.3 ng/l) over a period of three months. These peaks originated from runoff from cattle pasture (Fig. 5). Later peaks consisted only of testosterone that was moderately associated with sulfate (r²=0.53, P<0.05) and somewhat associated with total phosphorus (r²=0.49, P<0.1) indicating that the origin was leaching from the sulfate rich peat soil typical of the Hula Valley (Fig. 6). Testosterone in this catchment area was therefore due initially to surface runoff from cattle pasture and then as discharge from the soil.  The testosterone pulses after each rain event were more pronounced at the Joseph Bridge than at the Huri Bridge (Fig. 7).  Ethinylestradiol was consistently above 1 ng/l at three sites, two of which were known to receive sewage effluent and one had a high ammonia (>1.2 mg/l) and bacteria count (>50,000 fecal coli/100 ml), probably originating from a leaky sewage holding pond. There was no correlation (r<0.2) with the amount of testosterone, ethinylestradiol, estrogen or estradiol present in the samples with each other. However in the few sites (5/28) in which estriol was above 1 ng/l, ethinylestradiol was also above 1 ng/l (15/28).  A more detailed report on the results for 2001/2002 and 2002/2003 hydrological years can be found in Shore et al., 2004.
                       
                               

                     Ng steroid/l        

Figure 4.  Hormonal profile of runoff from cattle pasture.  

Figure 5.  Testosterone and sulfate concentrations in the runoff in the Hula Valley after rain events in the 2001/2002 hydrological year.

Table 1. Representative testosterone and ethinylestradiol concentrations at 14 sites on the Eastern side of the Hula Valley.

Site no.	Site name	Testosterone ng/l	Ethinylestradiol ng/l		Comments
1	Dan Tributary	1.1	1.3
2	Hermon Tributary	0.9	1.1	Some raw sewage
3	Sneir tributary	1.4	0.7
4	Hyun Creek	1.1	0.9
5	Joseph bridge	2.0	0.5	Confluence of sites 1,2,3,4
6	Kalil Canal	1.3	0.8
7	Yardonin Creek	2.1	1.2
8	Gonen Canal	2.2	1.6	High ammonia and fecal coli
9	Bitachon Canal	1.4	1.3
10	Lehavot Bridge	1.2	0.7	River below sites 6,7
11	Green Bridge	1.2	0.5	River below site 8
12	Hardale Bridge	1.3	0.7	River below site 8
13	Western Canal	2.1	1.2	Some treated urban effluent
14	Pkak Bridge	1.5	0.8	River below Confluence of sites 12,13 and 9.
Mean±SD (no. of samplings)		1.5±0.4 (125)	0.9±0.3 (112)

                   ng/l testosterone

Figure 6. Testosterone concentrations at the Joseph and Huri Bridges over the Jordan River. Samples were taken after each rain event of great than 30 mm/72 hr and during the dry season during which there is no precipitation (Days 225-284). Numbers on the x axis for individual rain events are the days numbered from 26.10.2001 which marked the initial rainfall.

Western part of the Hula Valley

Catchment  Streams:

Two subcatchments (Fig. 3) draining primarily cattle pastures were sampled at 15 sites on two dates following rain events representing 2 subcatchments draining primary cattle pasture. The amount of rain on the two dates sample was 21 and 36 mm/72 h respectively. On the date of the first sampling there were major spills of secondary effluent from sewage ponds.  Streams were considered contaminated with spillage effluent as the ethinylestradiol was detectable (>0.3 ng/l). In the contaminated streams, androstenedione and estrogen were significantly elevated (P<0.05) above the same streams without contamination while testosterone concentrations were not (Fig. 7). 

 Fig. 7.   Testosterone, androstenedione, estrogen, ethinylestradiol concentrations in ng/L determine in samples of water from 15 sites sampled after two rain events in two subcatchments draining primarily cattle pasture.  On one of the two dates, there were major spills of sewage water into the catchments (spillage).  Values are means ±SD.

 Studies of cattle pasture

First we measured the manure content for testosterone and androstenedione by collecting samples at 10 sites, isolated them from the cattle, and sampled the manure piles at one month and three months later. It was apparent that both compounds were excreted in about equal amounts and that there was little change over the three month period (Table 2).

Table 2.  Androstenedione (A) and testosterone (T) in cattle manure, 1 mo. and 3 mo. after being on pasture.  Ten sites were sampled on each occasion. Values are means ± SD of 10 determinations.

	mg/kg (after 1 mo.)	mg/kg (after 3 mo.)	1 mo. vs. 3 mo.
T	15.9±2.4	25.2±6.9	P<0.01
A	13.9±2.1	17.8±2.6	P<0.01
T vs. A	P<0.03	P<0.01

We then measured the amount of hormones in the soil after three months of grazing (Table 3) as well as the concentrations of testosterone and androstenedione in the runoff at the edge of the field (Table 4). It was apparent that androstenedione did not penetrate the soil as did testosterone.

 

Table 3.  Samples from a pool draining the same field as in Table 2.  Samples were taken after three rain events of at least 70 mm in the proceeding 72 h and tested for testosterone and androstenedione. Each point is a single observation done in duplicate.

Rain event	Testosterone (ng/L)	Androstenedione (ng/L)
08.01.04   6:40 10:40 11:50	2.0 2.1 1.7	2.1 1.8 1.6
24.01.04	0.7	1.8
27.01.04	0.7	2.2

Table 4. Testosterone and androstenedione extracted from soil samples at 5, 15, and 30 cm in a field use for cattle pasture, 4, 5 and 6 mo. after introduction of the cattle. Each value represents a single determination done in duplicate.

Soil depth	4 mo.	5 mo.	6 mo.
Testosterone  (ng/Kg)
5 cm	280	250	228
15 cm	130	86	56
30 cm	124	182	40
Androstenedione (ng/Kg)
5 cm	26	50	28
15 cm	14	40	8
30 cm	10	18	12

Studies of the effects on fish

Catchment streams: A survey of the 14 species of fish present in the Dan and Sneir rivers and four other sites in the Hula Valley catchment did not show any evidence of skewed sex ratio (Kroton, 2004}.  However, in a nearby stream (Nachal Kibutzim) which had a rich variety of species (13 species), most species were identified as having a skewed sex ratio. Of particular concern was Acanthobrama lissneri whose habitat is unique to the area.  In this stream river, measurable concentrations of ethinylestradiol (0.6-0.8 ng/l) were found.   The source of the ethinylestradiol appeared to be from the large number of swimmers in the small stream rather than contamination with sewage effluent.

Aquaculture reservoir

Hormone profiles: In the aquaculture pond, the concentration of steroidal hormones in the pond water increased with maturation (Table 5).  In the first months the amount of testosterone was comparable to that of androstenedione, but at the time the spawning (8 months), androstenedione was three fold higher than testosterone.

Prior to introduction of the fish, the reservoir was found to contain appreciable amounts of ethinylestradiol, medroxyprogesterone (both components of contraceptive pills); and benzodiazepines but was negative for barbiturates (Table 6.). After three months in the presence of the fish the levels of these compounds were essential non-detectable (<0.5 ng/l).  In contrast, in a control reservoir without fish, the level of medroxyprogesterone and ethinylestradiol remained between 1.3 and 1.7 ng/l during the same time period. 

Fish gonads: There were three species of fish in the reservoir, Oreochromis aureus (Tilapia), Cyprinus carpio (carp), Mugil cephalous (mullet).  The Tilapia had been treated with methyltestosterone so no ovaries were expected. However the fish should have had masculine gonads, which were absent.  The mullet usually take two years or three years to mature so no gonads were expected in the mullet. However the carp should have had gonads weighing about 800 grams at eight months and the ratio of the GSI should have been in the order of  4 %.  We found that the GSI in the females was between 0.4 to 3% and in the males, the testes were rudimentary.  Histological examination of the ovaries indicated the ovaries were fully matured and there was no evidence of intersex.  Some of the male carp were transferred in Nov. 2006 to a pond.  In the second year, the same males were placed in same water without exposure sewage effluent.  All of the nine male fish examined developed normal sized gonads after 4 months of growth.   

Table 5.  Steroid hormones in an aquaculture pond containing about 400,000 fish.  The pond was sampled 1, 3 and 8 months after introduction of the hatchlings.  Values represent the mean of three samplings at 1, 3 and 6 meters.

Months	Testosterone ng/l	Estrogen  ng/l	Androstenedione ng/l
1	1.62	0.97	2.37
3	3.75	2.20	4.67
8	5.73	7.07	18.23

Table 6:  Pharmaceutical concentrations in ng/l in reservoir water.

	Ethinyl-estradiol	Medroxy-progesterone	Benzo-diazepenes
Before introduction  of fish	1.43	0.5	Weak
One month after	1.20	0.5	Negative
Three months after	<0.5	<0.5	Negative
Eight months after	0.6	<0.5	Negative

Discussion

The major observations on steroidal hormones in the Hula Valley are summarized in Figure 8.

Figure 8.  Summary of observations on source, transport and effects

 of natural and synthetic hormones in the Hula Valley

In the Jordan River and its tributaries, hormone pulses were seen after rain events, particularly in the early part of a hydrological season.  In the dry season, hormone levels were essentially undetectable. At the start of the hydrological season, testosterone, androstenedione and estrogen pulses were observed due to runoff from cattle grazed fields and effluent from fishponds. These initial testosterone and androstenedione pulses dissipate over a three and five month period respectively. The absence of an estrogen pulse is due to estrogen binds tightly to the soil. The longer androstenedione pulse is due to testosterone penetrating the soil while the androstenedione remains on the surface but unlike estrogen is not tightly bound to the soil. The rest of the season is characterize by testosterone pulses which presumably come from soil washout as the pulses correlate with sulfate and phosphorus which are released from peat soils during the same rain events.  

These data suggest the hypothesis that there are four patterns of testosterone transport in the environment: (1) Testosterone associated with estrone, ethinylestradiol and estriol which are characteristic of sewage effluent; (2) Testosterone associated with estrone and estradiol which is characteristic of runoff from cattle pasture and manure fertilized fields; and (3) Testosterone alone, characteristic of leaching from soil and baseflow and (4) androstenedione in a much higher concentration in relation to testosterone which is characteristic of fishpond effluent (Kolodziej et al., 2004).

 Aquaculture

Testosterone, estrogen and androstenedione began rising the fourth month of development.  Androstenedione reached a maximum at the time of spawning.   The high androstenedione may be related to the large increase in androstenedione characteristic of spawning carp species (Sorensen et al., 2005). 

The male carp in the first year exposed to the ethinylestradiol in the range of 1.2-1.4 ng/l and did not develop gonads.  On the other hand, the female carp developed normal ovaries. This indicated that the effect was from an endocrine disruptor like ethinylestradiol which is well documented to affect male gonads in such low concentrations (Christiansen et al., 2002). However this would be the first report in non-laboratory setting that complete failure to develop testes was observed, apparently with just three months exposure to the compound.  Interestingly, in the second year, when the same fish were grown without exposure to ethinylestradiol, the gonads developed normally.  The effect therefore was not permanent. 

References

Barel-Cohen, K., Shore, L.S., Shemesh, M., Wenzel, A., Mueller, J. & Kronfeld-Schor, N. 2005. Monitoring of natural and synthetic hormones in a polluted river.  Journal of Environmental Management 78:16-23.

Casey, F.X.M., Hakk, H. & Simunek, J. & Larsen, G.L.  2004. Fate and transport of testosterone in agricultural soils. Environmental Science and Technology 38:790-798.

Colucci, M.S., Bork H. & Topp, E., 2001. Persistence of estrogenic hormones in agricultural soils: I. 17β-estradiol and estrone. Journal of Environmental Quality 30:2070-2076.

Christiansen, L.B., Winther-Nielsen, M. & Helwig C. 2002. Feminisation of fish. The effect of estrogenic compounds and their fate in sewage treatment plants and nature, Environmental Project No. 729. Danish Environmental Protection Agency. Available at: http://www.mst.dk/udgiv/publications/2002/87-7972-305-5/html/default_eng.htm

Degani, G., Boker, R., Jackson K., 1996. Growth hormone, gonadal development and steroid levels in female carp (Cyprinus carpio). Comp. Biochem. Physiol. C: Pharmacol Toxicol Endocrinol 115:133-140

Degani, G., Boker, R., Jackson K., 1998. Growth hormone, sexual maturity and steroids in male carp (Cyprinus carpio). Comp. Biochem. Physiol. C: Pharmacol Toxicol Endocrinol 120:433-430.
Gafny, S., Gasith, A. & Wise, G.S., 1999 - The Enan reservoir. In: Reservoirs for wastewater storage and reuse. Ecology, performance and engineering design. (eds M. Juanico and I. Dor). Springer, Environmental Science Series, Berlin, pp. 369-387.

Jürgens, M.D., Holthaus, K.I.E., Johnson, A.C., Smith, J.J.L., Hetheridge, M. & Williams, R.J., 2002. The potential for estradiol and ethinylestradiol degradation in English rivers. Environmental Toxicology & Chemistry 21:480-488.

Kolodziej, E.P., Harter, T. & Sedlak, D.L. 2004. Dairy wastewater, aquaculture, and spawning fish as sources of steroid hormones in the aquatic environment. Environmental Science & Technology 38:6377-6384.

Kroton, Y.  2004. Biodiversity and community structure of Jordan system fishes and their relationship to the habitat structure and anthropogenic impact. Thesis M.Sc, Dept. of Zoology, Tel Aviv University.

Lintelmann, L., Katayama, A., Kurihara, N., Shore, L. & Wenzel, A., 2003. Endocrine disruptors in the environment (IUPAC Technical Report). Pure and Applied Chemistry 75:631-681.

Segner, H., Caroll, K., Fenske, M., Janssen, C.R., Maack, G., Pascoe, D., Shהfers, C., Vandenbergh, G.F., Watts M. & Wenzel A., 2003. Identification of endocrine-disrupting effects in aquatic vertebrates and invertebrates: report from the European IDEA project. Ecotoxicological and Environmental Safety 54:302-314.

Shore, L.S., Correll D. & Chakroborty P.K., 1995a. Fertilization of fields with chicken manure is a source of estrogens in small streams, in Steele K., ed., Animal Waste and the Land-Water Interface, Lewis Publishers, Boca Raton, Florida, pp. 49-56.

Shore, L.S., Gurevich M. & Shemesh M., 1993. Estrogen as an environmental pollutant. Bull. Environmental Contamination and Toxicology 51: 361-366.

Shore, L.S., Hall D.W. & Shemesh, M., 1997. Estrogen and testosterone in ground water in the Chesapeake Bay Watershed. Dahlia Greidinger Inter. Symp. on Fertilization and the Environment, pp. 250-255, Technion, Haifa, Israel.

Shore, L.S., Harel‑Markowitz E, Gurevich M, Shemesh M.  Factors affecting the concentration of testosterone in poultry litter.  Environ Sci Health Part A.: 1993; A28:1737-1749.

Shore, L.S, Kapulnik, Y., Ben-Dov, B., Fridman, Y., Wininger, S. & Shemesh, M. 1992. Effects of estrone and 17-estradiol on vegetative growth of Medicago sativa. Physiologia Plantarium 84: 217-222.

Shore, L.S., Kapulnik, Y., Gurevich, M., Wininger, S., Badamy, H. & Shemesh, M., 1995b. Induction of phytoestrogens production in Medicago sativa leaves by irrigation with sewage water. Environmental & Experimental Botany 35:363-369.
Shore,L.S., Reichman, O., Shemesh, M., Wenzel, A.&  Litaor, M.I., 2004. Washout of accumulated testosterone in a watershed.  Science of the Total Environment 332:193-202

Shore, L.S. & Shemesh, M., 2003. Naturally Produced Steroid Hormones and their Release into the Environment. Pure and Applied Chemistry 75:1859–1871.

Sorensen P.W., Pinillos, M. & Scott, A.P.  2005. Sexually mature male goldfish release large quantities of androstenedione into the water where it functions as a pheromone. General & Comparative Endocrinology 140:164-75.

Wenzel, A., Kuechler, Th. & Mueller, J., 1998,  Konzentrationen oestrogen wirksamer Substanzen in Umweltmedien. Report. Project sponsored by the German Environmental Protection Agency; Project No 216 02 011/11 (In German).

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Ph.D. with P.H.D. (Piled High and Deep)

  

endocrinology 2011 eyes

Canis domesticus

The effect of neutering on tear production in dogs

 R Ofri¹, R Ckalifa¹, T Bdolah-Abram¹, L Shore²
Koret School of Veterinary Medicine, Hebrew University of Jerusalem, Israel¹;
Department of Endocrinology, Kimron Veterinary Institute, Ministry of Agriculture, Israel².

 Purpose: There are conflicting reports regarding gender predisposition to keratoconjunctivitis sicca (KCS), with some authors demonstrating the disease to be more prevalent in female dogs. While this finding is disputed by others, there appears to be a consensus that neutered dogs, of either gender, are predisposed to KCS, probably due to decline of sex hormones regulating tear production. Our aim was to evaluate tear production in dogs prior to, and following, neutering and to correlate it with levels of sex hormones. Methods: Study animals included 25 healthy dogs (14 females, 11 males) presented for elective neutering. Schirmer Tear Test I (STT) was performed, and blood samples for progesterone, estrogen and testosterone assays were withdrawn, on days 0 and 14. Fourteen dogs (8 females, 6 males) were re-evaluated 6 months later. Results: Mean±sd STT results on days 0, 14 and 180 were 19.7±4.4, 19.0±3.6 and 20.3±6.9 mm/min, respectively. There were no significant differences between the three readings (P=0.65), or between males and females (P=0.40). Nor was there a significant decrease in STT over time in either neutered males or females (P=0.99). No significant correlation was found between STT and any of the three hormones assayed (P>0.10). Conclusions. We believe this small scale, pilot study is the first one to measure STT over time in the same dog, prior to and following neutering. While the study needs to be repeated with a larger sample, it seems that in dogs, unlike other species, neutering has an insignificant effect on tear production.

European College of Veterinary Ophthalmologists (2011)


Capra nubiana  (pictures courtesy of Ron Ofri)
Ibex  Intraocular pressure 

Panthera leo

Ofri, R., Shore, L.S., Kass, P.H. and Horowitz, L.H. (1999). The effect of elevated progesterone levels on intraocular pressure in lions (Panthera leo). Research in Veterinary Science 67:121-123

animal endo 2011 zearalenone, phytoestrogens

Bos bovis

The effects of Zearalenone on ovarian function in dairy cows

Uzi Moallem, Moshe Kaim  Volcani Institute

It is well documented that compounds similar to estrogen in containing a phenol group can cause disturbances in the reproductive organs of various species. These estrogen-mimetics may exert their effects by two major actions: 1) binding to the estrogen receptor as an agonist; 2) binding to the estrogen receptor and preventing estrogen action (antagonist). Zearalenone is a well-known estrogen-mimetic which binds to the estrogen receptor and can decrease reproductive function in pigs, cattle and chickens.  Zearalenone has been reported to decrease conception rates and increase abortion incidence in cattle.  Similarly zearalenone is suspected to cause cyst formation in the bovine ovaries. One report was that in heifers fed 12.5 ppm of zearalenone/day, there was a reduction in the inception rate. A similar problem was reported in a milk herd which was feed with moldy hay with high concentration of zearalenone. The goal of the present experiment was to examine the effect of a known amount of zearalenone on the bovine reproductive system.  The experiment was undertaken using the experimental barn of the Volcani Institute using 12 cows which had at least one milking cycle.  Six cows were given feed containing 5 mg/day of zearalenone (Sigma) while six other cows served as a control. The zearalenone was mixed with 20 grams of ground corn and feed to the individual cows. The cows were synchronized and the ovaries were scanned for 2 consecutive cycles.  The scans recorded the follicles, corpora lutea and any ovarian abnormality. During the third cycle, follicular fluid was suctioned form the pre-ovulatory follicles. Samples of blood were taken at least once a week and more frequently in the peri-ovulatory period for estradiol determinations. No difference was observed between the groups in the size of small and intermediate follicles but the number of follicle greater than 15 mm was higher in the treated group compared to controls. Similarly the diameter and volume of pre-ovulatory follicles was greater in the treated over the control but this did not reach significance.  Analysis of the follicular fluid did not find significant differences in progesterone, androstenedione or estradiol concentrations. However the total estradiol/follicle was twofold higher in the treated compared to control (P<0.1). Similar observations were made for androstenedione and progesterone. The concentration of progesterone in the plasma on day 14 of the cycle was lower in the zearalenone treated than control (3.25 vs 6.55 ng/ml, respectively).  Examination of plasma estradiol in the peri-ovulatory showed no differences between the groups.  The data suggest that there were differences in steroid hormone production in the ovaries feed 5 mg/day zearalenone.  This was reflected in lower plasma progesterone but not plasma estradiol. Because of the high cost of the material and the intensive work schedule required, this experiment was limited to only 12 cows.  We suggest that if a larger group of cows were used, a statistical robust effect would be obtained for most of the parameters reported here.

Hebrew report  http://www.halavi.org.il/info/research/1/362-0159-08.htm

Zearalenone in the feed is sometimes associated with abortion in cows. In this unusual case, there were nearly 50 abortions in the time observed.  The peak in abortions was in second trimester.

Phytoestrogens - effects on fertility

Structure of the phytoestrogens 

Genistein and daidzein are converted to the more potent phytoestrogen equol by the intestinal bacteria.

Structure of the phytoestrogens

Plasma estrogen concentrations in cows on day of insemination in a herd feed vetch and three months after the vetch was discontinued Winter 1996).  Higher plasma estrogen was associated with higher resorption rates.

Legumes and resorption in dairy cows

Fetal resorption percentages in cows fed legumes.

Vicia sativa  vetch

Vigna unguiculata  cow peas

Resorption was determined as described in Shore, L.S., Rios, C., Marcus, S., Bernstein, M. and Shemesh, M. (1998). Relationship between peripheral estrogen concentrations at insemination and subsequent fetal loss in cattle. Theriogenology 50:101-107.  Cows were fed 1 to 3 KG of legumes/dy.  The principle phytoestrogen in cowpeas is formonentin but the phytoestrogen in vetch  could not be identified.

Here is a chart indicating that feeding vetch (a legume) to bull calves results in smaller scrotal circumference.  

Environmental 2011

Bos bovis

Soil bacteria which utilize aromatic carboxylic acids

Hermann, G., Shore, L., Steinberger, Y. (2011) Effects of cattle-lagoon slurry on a soil microbial community can be observed until depths of 50 m. Applied Soil Ecology 49 (2011): 32– 39

The large amount of effluent generated by concentrated animal feeding operations (CAFOs) has raised concerns about contamination of groundwater and pollution of streams by compounds that penetrate the vadose zone. However, the possibility that a microbial community in the vadose zone under cattle- slurry lagoons (CSLs) may also be affected has not been considered. In the present study, we investigated the influence of long-term (30 years) accumulation of cattle slurry on the vertical distribution of a soil microbial community (microbial biomass [MB], CO2 evolution, substrate utilization ability), until a 50-m depth, compared to a control site. Total soluble nitrogen (TSN) was found to be elevated fourfold, and MB was found to be threefold higher under the CSL compared to a control site. In general, the increase in MB is associated with higher soil moisture and higher nitrogen content. Substrate utilization ability was found to be significantly higher in a CSL in comparison to the control site. At the CSL site, a higher utilization of aromatic carboxylic acids typical of cattle slurry was obtained in the deeper soil layers (7–30 m), indicating a degree of microbial adaptation even at these depths. The soil layers under the CSL were more dynamic as the microbial functional diversity was significantly different between the layers, while no such difference was seen at the reference site. Our results, therefore, suggest that the infiltration of cattle slurry can affect the microbial community throughout the vadose zone. We also suggest that activity of the microbial community, as characterized by its substrate utilization ability, can be a bioindicator for anthropogenic activities and environmental changes even at depths below the rhizosphere (30 cm).

animal endo 2011 nematodes

Nematoda

Soil inhabiting nematodes

 Bacterivores

Acrobeloides

Cephalobus

Fungivores

Aphelenchus

Hu, C., Hermann, G., Pen-Mouratov, S., Shore, L., Steinberger, Y. (2011) Mammalian steroid hormones can reduce abundance and affect the sex ratio in a soil nematode community.  Agriculture, Ecosystems and Environment 142 (2011): 275– 279.

The effect of mammalian steroid hormones (estradiol, estrone, progesterone, and testosterone) in environmentally relevant concentrations on a soil free-living nematode community was examined. Steroids were applied in 2.5 L water on 0.25 m2 plots in triplicate, and comparison was made with plots receiving water alone. Soil samples were taken from the 0 to 5, 5 to 10, and 10 to 20-cm soil layers. The soil free- living nematode populations were examined at zero time, 12 h, 72 h, 96 h, 120 h, and 25 d post-treatment. Testosterone, progesterone, and estrone were persistent in the soil while the estradiol rapidly dissipated. The total number of nematodes was decreased by the presence of testosterone, progesterone, or estrone but not estradiol. Testosterone, progesterone, and estrone increased the number of males in relationship to females (60:40) compared to the control and estradiol-treated plots (50:50). The presence of steroids had no consistent effect on the distribution of bacteria-feeders, plant-feeders, and omnivore-predators. We conclude that the addition of steroid hormones in the soil can reduce abundance and change the sex ratio in a free-living nematode community. This would be the first demonstration of an effect of an endocrine-active agent excreted by mammals on a free-moving terrestrial organism.

 

Diagram of head regions of a herbivore (left) and nematode bacterivore (right). In the herbivore, the mouthpart is modified into a stylet for puncturing plant cells. In the bacterivore, the mouth or stoma is a hollow tube. (from http://entomology.ifas.ufl.edu/creatures/nematode/soil_nematode.htm)

animal endocrinology 2011

1.           Ptyodactylus guttatus

Guy Sion, Hebrew University, Dept. of Ecology, Evolution & Behavior

2.           Oculina patagonica

Rachel Armoza- Zvulani, Tel Aviv University, Dept. of Zoology

3.           Mandrillus sphinx

Yael Tal-Nissim, Hebrew University Vet. School

This is about lizards not wizards.

(from Wizards of Beverly Place)

Ptyodactylus guttatus

Aggressive geckos use their left eye as this information goes directly to the aggressive right brain.  We are investigating if this agression is associated with higher hormone levels.

Oculina patagonica

In this study we compared reproductive characteristics and steroid hormone levels in the non-indigenous scleractinian coral, Oculina patagonica, inhabiting contaminated vs. uncontaminated reference sites along the Israeli Mediterranean coast. Our results indicate significantly higher steroid levels in both seawater and coral tissue samples from contaminated sites as compared to reference sites, suggesting that corals tend to accumulate steroids from the surrounding waters. Despite their higher steroid levels, corals from the contaminated sites showed reproductive potential comparable to those of the reference sites. Interestingly, a clear distinction could be seen between corals exposed to pollution for long vs. short durations, with the latter showing a failure to complete gametogenesis. This suggests that reproduction in O. patagonica is susceptible to acute rather than chronic stress. The involvement of acclimatization/ adaptation processes may explain this species tolerance, and may reflect the ability of O. patagonica to successfully invade new areas in the Mediterranean Sea. 

Armoza-Zvuloni, R., Kramarksky-Winter, E., Rosenfeld, H., Shore, L. S., Segal, R., Sharon, D., Loya, Y. (2012). Reproductive characteristics and steroid levels in the scleractinian coral Oculina patagonica inhabiting contaminated sites along the Israeli Mediterranean coast.  Marine Pollution Bulletin 64 (8): 1556-1563

http://dx.doi.org/10.1016/j.marpolbul.2012.05.020

Mandrillus sphinx

Although mandrils are an endangered species in the wild, they breed too well in captivity.  In order to determine the best contraceptive protocol using a GnRH agonist, fecal steroids were determined before and after implantation of the agonist.