Halacha and Habitat

This was a draft for an article for a journal on Judaism and the environment which ceased to exist. 

Environmental changes as reflected in halachic responsa.
Aryeh Shore

“is it possible that cows will no longer thresh grain or plow fields?.. is it possible that the shore will no longer hold back the sea?” Siphra, Haazinu.

The passage of time makes it difficult sometimes to understand the milieu in which rabbinical responsa were written.   Facts that were readily apparent to the authors and therefore taken for granted become an impediment for understanding these responsa in later generations.  The present paper addresses the ecological background of several responsa, e.g. extinction of species, beaching of whales, habitats and availability of natural resources.  These will be considered in the framework of the historical background of the responsa.

1.    Extinction of Species – Bos primigenius

2.    Beaching of whales

3.    Habitat - Acipenser Sturio Acipenser ruthenus

4.    Introduction of new species,

5.    Changes in availability of a natural resource – the availability of bitumen from the Dead Sea and mountain springs (mumia).

Extinction of animals

That an animal may exist for only a certain period of history is mentioned in the Talmudic discussion of the Tachash (Exodus 35:7, Shabbat 28b, Midrash Tanchuma VI). However Rabbi Yisrael Lipschulz  (1781-1860;Tiferet Yisrael, Killaim 8,5) is apparently the first halachic authority to suggest that animals mentioned in the Mishna may now be extinct “like the mammoth”.

Bovis primigenius

The ancestor of all domestic cattle is Bovis primigenius or the Auroch.  There were three lines of this animal but only the European Auroch (Bovis Uris) survived till modern times. Although it seems certain that the bible mentions the Auroch or wild ox, it is not clear which of the terms used: Re’em (JPS Bible) or Tao (Septugent, Targum Yonaton-Forest Ox), both which may refer to the Auroch.

      The wild ox described by Rashi in Sanhedrin (28a), which he presumably saw, could be domesticated. This would be in contrast to the alternative European bison or buffalo (Bison Bonasus) (an endangered species), which was not domesticated. Since the Bovis Uris described by Julius Caesar could be also domesticated (De belli Gallici, 6.XXVIII. De uris, Sed adsuescere ad homines et mansuefieri ne parvuli quidem excepti possunt ) it would appear that the extinction of this animal which occured in the 17^th century (http://en.wikipedia.org/wiki/Aurochs) was a source of confusion to later generations which did not see this animal. An alternative hypothesis is that the source of confusion stems from the geographical habitat of the water buffalo (Kashrut of Exotic Animals: The Buffalo, Ari Z. Zivotofsky The Journal of Halacha and Contemporary Society, Fall 1999/Sukkot 5760, Number XXXVIII).

 Beached whales

Whaling is mentioned only once in halachic sources - in the French Tosephot (12th Century) on Bechorot 7b. “It is permitted to eat the murina (eels) [hareng] found in the stomachs of whales (balaena).”
     Although there was a thriving whaling industry in the Bay of Biscay in the 12th century, the slow moving whale, Eubalina galcialis (an endangered species), that was hunted by the Basques, ingests primarily cephalopods.  It is more likely what is discussed is a beached whale which would be familiar to the Provencal Tosephot.  Beached whales were common along the Provencal coast and the stomach contents could arouse great interest in the local population as described for a 17^th century beaching of a roqual (Zoologische Einblattdrucke und Flugschriften vor 1800 [Zoological broadsides and pamphlets prior to 1800]. Vol. 4 (of 5): Wale, Sirenen, Elefanten. Stuttgart: Anton Hiersemann Verlag, 2002. 402 pp.).  There is evidence that the roqual does ingest herring (Do minke whales [Baleaenoptera acutorostrata] exhibit particular prey preferences? Skaug HJ, Gjusaeter J, Haug T, Nilseen KT, Linstrom U., J. Northw. Atl. Fish. Sci. 22:91-104, 1997) so herring (hareng) is probably the correct text version.

Habitat

As mentioned above for water buffalo, the geographical distribution of animals which were seen by poskim in some areas but absent in others, could lead to confusion in the responsa of later authorities. A good example is a core responsa on the discussion of the kashrut of fish (Nodeh B’yehuda, Mahadarah 2, 28,29) by R. Shmuel Landau (d. 1834), given in 1822 in Prague. The question asked was whether the sterlet was the esturgeon mentioned in earlier authorities and not the sturgeon.  However there were other factors which determined the response such as the Haskala movement and Sabbatism.

   The North Atlantic sturgeon was fished from earliest times to the end of the 19^th century. During that time it was found along the Mediterranean coasts of Italy and Spain as well as the North Atlantic but due to the massive overfishing of the 19^th century today it is an endangered species confined to the Baltic Sea. The "real" European Sturgeon Acipenser Sturio, der Stoer, up to 4.5 m long, was found regularly until the end of the 19th century in the Atlantic (including North and Baltic Seas) catchment basin. It is rarely found in the Black Sea catchment. But there are many other sturgeon species which are not always accurately determined and named by laymen. The Ruthenian Sturgeon, A. ruthenus, Sterlet (= small sturgeon, a diminutive in German) or sterljad inhabits the effluents to the Black Sea. Jews in Central Europe more frequently met A. sturio. In Eastern Europe the Jews would often see A. ruthenus and other species as Huso huso, A. nudiventris, A. gueldenstaedti etc. Sturgeon was traded intensely, at the beginning not mainly for caviar, but for its fresh or salted meat (The sturgeon [Acipenser sturio L., 1758] in Europe, Z. Okologie u. Naturschutz 6:129-135,1997; Kinzelbach, R. (1994): Ein weiterer alter Nachweis des Sterlet, Acipenser ruthenus, in der worttembergischen Donau. - In: Biologie der Donau. Limnologie aktuell 2: 281-291, Stuttgart.)

   Both Rabbenu Tam (France 12^th century) and the Ramban (Spain 13^th century) approved the use of a fish called esturgeon (Shemesh Tzedakah, 1742 Venice, YD 14). In order to prove that esturgeon was the common sturgeon, Acipenser Sturio, R. Landau used a book on fish written by a Jewish physician from the Jewish community of Berlin. From R. Landau's description, this book was Allgemeine Naturgeschichte Der Fische by Marcus Eliaser Bloch (1723-1799), a Jewish physician and ichthyologist associated with the Haskala movement. R. Landau was particularly impressed with the list of names for sturgeon  given by Bloch in different languages, e.g. esturgeon (Fr.), storione (It.); porcelleto (young sturgeon, It.). The book by Bloch is still reprinted today, albeit more for its artistic depictions than its scientific interest.  R. Landau also uses the book to prove that the sterlet is Acipenser ruthenus based on size and picture.  This would appear to be the first time a modern natural history textbook is cited in a responsa. However he was not alone in using such sources as Landau’s contemporary, R. Y. Lipshultz quoted Die Urwelt by H.F. Link in his commentary on the Mishna (Shore A, 1997).

Acipenser Huso  (Huso huso)                                                    can be purchased at www.oldprintshop.com        

Acipenser Ruthenus                               Gordon Edmondson Sturgeon Collection                                                                         

Acipenser Sturio

  
       Other historical factors can be seen in R. Landau's discussion of the use of sturgeon by the Turkish Jews. (It is not clear to which source he is referring. The Jews of Izmir commonly ate honeyed ovaries but this was mullet roe, [Kinselbach, personal communication]. The objection from the opponents of sturgeon was that there were Sabbateans in Turkey and therefore information from that country was suspect. R. Landau rejected this criticism.

Introduction of new species

Introduction of new species naturally received a lot of attention in the responsa as questions of whether it should be considered either bush or tree or should be considered  fruit or vegetable needed to be decided. Similarly new animals required determination if they were kosher or not.

There were two main periods of halachic discussion:

1. Introduction of new species with rise of the Islamic empire. This resulted in introduction of new species from southeast Asia such as eggplant (Solanum melogena) and sugar cane (Saccahrum officinarum). These have been discussed by Amir (Z. Amir, Agronomic innovations as reflected in medieval biblical commentators and halachic authorities, BDD 3:87-96, 1196, in Hebrew).

2.  Introduction of new species with the discovery of the new world. The impact of these plants and animals on halachic responsa continues to the present day and are addressed in nearly every major halachic work (Shore A.  The use of scientific sources by rabbinical authorities to determine the nature of animal species. Korot (Heb. Univ.) 12 (1996-1997): 7-15; Zivotofsky 1999 op cit; Zivotofsky Is Turkey Kosher? The Journal of Halacha and Contemporary Society, 1998, Vol. 35:79-110.).

Changes in availability of natural resources – mummia

The selling by Jewish merchants of mummy, material presumably derived from Egyptian mummies, presented Halachic problems to several Poskim (R. David ibn Zimra [HaRadbaz, Egypt, 1480-1573], R. Avraham Halevi [Egypt 1650-1710?]; R. Yehuda Rosanes [Constantanople, 1657-1727]. The material is described as small pieces of resilient bones or a tarry dust. The main points raised against the practice were (1) In the case of the merchants being Kohanim, they are not permitted to touch or carry parts of dead people, whether Jewish or not, less they become “tameh”; (2) It is forbidden to derive benefit from the dead and selling is considered a benefit; and (3) It is forbidden to sell food which is forbidden to eat and human flesh is forbidden to eat. These responsa have been dealt with extensively in a monograph by E. Reichman (The impact of medieval medicine on medical Halachah. In: Pioneers in Jewish Medical Ethics. Rosner F., ed., Jason Aronson, Northvale, NJ., 1997, p.27-52).

     The disturbing ethical aspects of selling mummy were not limited to the Jews. Ambrosie Pare (1510-1590) wrote “The ancients…did not contemplate such an abomination but were either thinking of the universal resurrection or of the memory of their dead parents or friends” (Discours de la mumie, Paris 1582). Pare also noted that Jews were widely involved in the selling of mummy.

     The background of these responsa goes back to events thousands of years before they were written.  The areas considered here are the medicinal history of mumia, methods of making mummies, and the Dead Sea bitumen industry. It is also hypothesized that the seismology of the Dead Sea played a role.

What is Mumia?

     The ancient Persians made medicinal use of a black, bituminous substance “mimia”, which oozed from a local mountain (Mumia naturalis persica). The current source of Mumyoe (Russian) is from the floors of mountain caves in Tajikistan. The substance is the digest of ancient flora at the crest of the mountains, which then drops to the base of the caves where it is collected. Although touted in Russia as a food additive with anabolic properties, in Israel it is sold for intestinal disorders, fragile bones, inflammation, chronic bronchitis, glucose regulation and increasing immunity (according to the package insert).

How the mumia became mummy.

The ancient Egyptian used a variety of resin and waxes in their preparation of mummies and these ingredients changed over the centuries.  The use of bitumen from the Dead Sea was limited to the period from 500 BC to the middle of the 1^st century (CE Buckley, S. A. & Evershed, R. P. Organic chemistry of embalming agents in Pharaonic and Graeco-Roman mummies. Nature, 413, 837 - 841, 2001; Ancient and modern medicinal application of Dead Sea asphalt (bitumen), Isr. J. Earth Sci: 48:301-308 1999).  This industry relied on blocks of bitumen rising from the depths of the Dead Sea as described by Hieronymus of Cardia in 312 BC (Diodorus Siculus, Library of History, trans. Oldfather D.H., Heineman, London, 1935).   The disappearance of bitumen at this time coincides with a period of seismic quiescence from 200 to 700 CE. Other historical factors such as high taxes by the Egyptians and the absorption of Egypt into the Roman Empire played a role in the demise of the bitumen industry. It is not known if or how much bitumen from the Dead Sea was exported to Egypt after this time, but a visitor to Egypt in 1200 mentions only the bitumen from mountain springs and that the mumia from mummies was only an alternative source (Abd al-Latif al Baghadidi, Book of Instruction and Admonition on Things and Events Seen in the Land of Egypt, trans. KZ Hafuth, JA Videan, IE Vidan (London:Allen and Unwin, 1964, f 41L,42R). This substitution for mumia naturalis may be due to increased demand as the prominent Persian physicians praised the properties of their locally produced material. However this material was very scarce as there was a royal monopoly (Al Biruni’s book on pharmacy and material medica, trans. H.M. Said, Hamdard Natl. Foundation, Karachi 1973, p. 311, s.v. mumya’u). Interestingly, each batch was tested for its potency to heal a fractured chicken leg (The book of curious and entertaining information The Lata’ if al-ma’arif of Tha’alibi. Trans. CE Bosworth, University Press, Edinburgh, 1968). (This would be a very early example of pre-testing a drug before distribution). However, scarcity alone may not have been the only reason. It was an accepted belief that juices from corresponding parts of a healthy body could be used for curing disease. Since the juice obtained from mummy pieces was extracted from every part of the body, Mumia factitia var. humana or kufr, could be a universal medicine against all diseases (Bosworth, C.E., Evan Donzel, W.P. Heinrichs, and C. Pellat, eds. The Encyclopaedia of Islam. New Edition. Leiden: E.J. Brill, 1992. s.v. "mumiya," by A.Dietrich).  It was during the 13^th century that medieval Europe learned that the ancient Egyptians used a bitumen mixture call “mumiya” to embalm their bodies and that by melting down this substance one could obtain an oil of great medicinal value. Beginning in that century, and throughout the Middle Ages, thousands of Egyptian mummies were exported from Alexandria to Europe by way of Marseilles - initially for recovery of the bitumen, later, as the effective ingredient was forgotten, to be simply ground to powder altogether. This powder called “mummy” was a standard apothecary ingredient. The use of ground mummy, with a wide variety of fake substitutes, continued to the early part of the 20^th century. Dried mummy (mummia) is still available today in the markets of Cairo (Z. Amir, personnel communication).

Discussion

   The premise of the present paper is to show that information from a variety of sources can increase the understanding of the responsa which contained details that were obvious at their writing but became obscure in later generations. There is an extensive literature on the scientific and biological background of the Bible and Talmud. The sources such as Ibn Sina and Ibn Rhodes which were used by the Sephardic physicians (e.g. Rambam and Ibn Ezra) have also received attention. However, similar information on the vast responsa literature and commentaries of the 11^th to the 19^th century is not readily available. For example, the controversy over the chicken without a heart is a core responsa for the use of science in determining halacha. However, it is not known where the experiment attributed by the Hacham Zvi to Galen is recorded nor how the response of the Halle Medical Society to R. Eybshutz appears in the German records. Similarly, what bestiary did the Bartinura use for his odd description of the leopard as a cross between the wild boar and lion in Avot 5, 20 which differs from the standard bestiary description of it as a cross between the lion (leo) and panther (pard). 

    In passing it should be noted that some of the species discussed here are endangered and some are on the verge of extinction. It may well be that in another hundred years scholars will have to consult natural history books to determine to which extinct species we are referring in modern responsa.

Note on Mummia from Moshe David Gaon (1889-1958). Emunot V'trufot Elil Bkerev Yehudei HaMizrach

When people became desperate they turned to "wise women" who were widely known.  They kept packages of lumps of mummia.  When the preparation was needed the material was mixed with honey and rolled into pills.   It was said that when the Moslem pilgrims went to the Haaj in Mecca, they would bring bones of dried corpses from the Arabian desert, and for a fat price they would sell them to the doctors to make the mummiyah.  More than once these disgusting pills were forced down my throat without an explanation. Later it was explained that it was against paleness in the face, weakness, shortness of breath, neglect of studies (attention deficit disorder??).  It was also a remedy against the Evil  Eye and to distance friends which the parent didn't like.

neuroscience 2012

Nephew's David's paper is now available online.

Nicaise, Charles, Hala, Tamara J., Frank, David M., Parker,

Jessica L., Authelet, Mich`ele, Leroy, Karelle, Brion, Jean-Pierre, Wright, Megan C.,

Lepore, Angelo C., Phrenic motor neuron degeneration compromises phrenic axonal circuitry

and diaphragm activity in a unilateral cervical contusion model of spinal cord

injury, Experimental Neurology (2012), doi: 10.1016/j.expneurol.2012.03.007

http://www.sciencedirect.com/science/article/pii/S0014488612001057

Abstract

Respiratory dysfunction is the leading cause of morbidity and mortality following traumatic spinal cord injury (SCI). Injuries targeting mid-cervical spinal cord regions affect the phrenic motor neuron pool that innervates the diaphragm, the primary respiratory muscle of inspiration. Contusion-type injury in the cervical spinal cord is one of the most common forms of human SCI; however, few studies have evaluated mid-cervical contusion in animal models or characterized consequent histopathological and functional effects of degeneration of phrenic motor neuron-diaphragm circuitry. In an attempt to target the phrenic motor neuron pool, two unilateral contusion injury paradigms were tested, a single injury at level C4 and a double injury both at levels C3 and C4, and animals were followed for up to 6 weeks post-injury. Both unilateral cervical injury paradigms are reproducible with no mortality or need for breathing assistance, and are accompanied by phrenic motor neuron loss, phrenic nerve axon degeneration, diaphragm atrophy, denervation and subsequent partial reinnervation at the diaphragm neuromuscular junction, changes in spontaneous diaphragm EMG recordings, and reduction in phrenic nerve compound muscle action potential amplitude. These findings demonstrate significant and chronically-persistent respiratory compromise following mid-cervical SCI due to phrenic motor neuron degeneration. These injury paradigms and accompanying analyses provide important tools both for understanding mechanisms of phrenic motor neuron and diaphragm pathology following SCI and for evaluating therapeutic strategies in clinically-relevant cervical SCI models/

Intact neuromuscular junction in phrenic muscle, characterized by: complete overlap of the pre-synaptic axon and pre-synaptic vesicles with post-synaptic acetylcholine receptors.

animal reproduction 2012

Sus
 Theriogenology available on line
http://authors.elsevier.com/TrackPaper.html?trk_article=THE12119&trk_surname =Karpas 

Marcus, S., Menda, A., Shore, L., Cohen, G., Atweh, E., Friedman, N., Karpas, Z. (2012) A novel method for the diagnosis of bacterial contamination in the anterior vagina of sows based on measurement of biogenic amines by ion mobility spectrometry: A field trial. Theriogenology (In press).

To determine if postpartum subclinical infection occurs in sows, a novel device was used to diagnose such bacterial contamination of the vagina. The device was based on the measurement of biogenic amines by ion mobility spectrometry (IMS). The device is portable and results are obtained within one minute. Vaginal swabs were taken from 449 sows prior to first- estrus insemination and 133 (29.6%) had elevated biogenic amines and were considered positives. Sixty one percent of the sows became pregnant following post weaning first estrus insemination. Positive scores had no apparent effect on fertility rate which was 64%. Of the sows that became pregnant 197 (69.1%) were diagnosed as “negative” and 88 sows (30.9%) were “positive”, of which thirty-seven sows received treatment with antibiotics and were termed "positive treated" .

The average live born piglets litter size of the “positives” was 10.02 which was significantly lower (P=0.031) than the "negative" sows (11.06) while “positive treated” sow average litter size was close to the "negative" (10.56). In conclusion, it was demonstrated that subclinical anterior- vaginal bacterial contamination in lactating sows about two weeks postpartum is a condition that affects sow litter number and could be determined by the measurement of vaginal biogenic amines with IMS.

sperm mediated transfer 2012

Eliane's thesis is available on line.

Manipulation of Sperm for Efficient Production of Transgenic Calves and Chicks.

Eliane Harel- Markowitz

http://shemer.mslib.huji.ac.il/dissertations/H/AGR/001520453.pdf

Abstract

The current method of micromanipulation used in domestic animals results in less than 1% transgenic animals, which makes it extremely difficult to produce transgenic calves and is completely unfeasible for producing transgenic chicks. The purpose of this work was to find a more efficient method for producing transgenic calves and chicks using a combination of two techniques, lipofection and restriction enzyme mediated insertion (REMI). Previously investigators were unable to produce transgenic chicks using lipofection alone, while injection of isolated sperm nuclei, which had been incubated with restriction enzyme, into oocytes had only been demonstrated in frogs. In this study, we demonstrated for the first time, that lipofection of both DNA and restriction enzyme could be used to successfully integrate DNA into sperm genome DNA and then used for routine AI to produce transgenic calves and chicks. First it was demonstrated using needle pricking and southern blot analysis of genomic DNA that the restriction enzyme opens up “hot”  spots in the sperm genomic DNA, producing sticky ends into which foreign DNA  could be inserted and integrated into the sperm genomic DNA rather than just free DNA floating in the fertilized egg. The “transgenic sperm” thus made were used in IVF and AI to produce embryos expressing a foreign DNA, GFP (green fluorescent protein). Using Not I and linearized pGFP lipofected to sperm for AI resulted with two calves which expressed the exogenous DNA in their lymphocytes as determined by (a) PCR and RT-PCR; (b) specific emission of green fluorescence by the GFP protein; (c) homology analysis between GFP DNA and PCR product DNA sequences and (d) Southern blot analysis.  Similarly in the chicken, linearized plasmid GFP sequences with the corresponding restriction enzyme (REMI) were lipofected into the sperm. The transfected sperm were then used for AI in hens and 90% (17/19) of the resultant chicks expressed the exogenous DNA in their lymphocytes as determined by  (a) PCR and RT-PCR; (b) specific emission of green fluorescence by the GFP and (c)  Southern blot analysis. A complete homology was found between the Jellyfish GFP DNA and a 313 bp PCR product of DNA from chick blood cells. The procedure was then tested with an additional construct, hFSH. The construct of hFSH consisted of both subunits, alpha and beta and the PCR product used primers for both alpha and beta subunit resulted with a PCR product of 584 bp which was unique to transgenic chickens. The procedure was then used to lipofect a construct of hFSH (Human Follicle Stimulating Hormone) into chicken sperm and used for AI. The resultant offspring were transgenic in at least three generations as determined by (1) measurement of hFSH protein in chicken blood using enzyme immunoassay (EIA) and RIA;  (2) RT-PCR and PCR; and (3) copy number.  We conclude that (1) lipofection of both DNA and restriction enzyme into sperm  (bovine and chicken) induces the integration of the DNA into the sperm genomic DNA; (2) lipofected sperm can be used in AI to produce a high percentage of transgenic calves and chicks; (3) The integrated gene is expressed in first, second and third generation; and (4) the method is not limited to specific genes. The technique of lipofection of DNA combined with REMI is therefore an efficient and stable method for producing transgenic domestic animals.  Efficient production of transgenic domestic animals could have major impact on gene therapy, improving livestock breeds and the production of valuable pharmaceuticals, e.g. hFSH, which could be extracted from eggs and milk.

The e-book Sperm-mediated Gene Transfer: Concepts and Controversies. (ed. K. Smith) is  online.

Sperm-Mediated Gene Transfer: Concepts and Controversies
DOI: 10.2174/97816080523701120101
eISBN: 978-1-60805-237-0, 2012
Editor: Kevin R. Smith  Abertay University

Manipulation of Sperm for Efficient Production of Transgenic Calves and Chicks Pp.92-102

Mordechai Shemesh, Laurence Shore, Yehuda Stram, Eliane Harel-Markowitz and Michael Gurevich

Introduction

Sperm-mediated gene transfer (SMGT) represents a novel set of technologies for animal (or in the future, human) genetic modification using the sperm as a vector, as opposed to more traditional established routes such as fertilized eggs or embryonic stem cells.

Studies of sperm-mediated gene transfer (SMGT) indicate that sperm cells possess the ability to be utilized as carriers of exogenous genetic sequences, offering the potential of a novel cost-effective route for germline genetic modification. The fate of transgenes borne by sperm cells has been inconsistent, and analysis of offspring from SMGT experiments has shown a mixed picture in terms of genomic integration of the transgene, suggesting an episomal mode of inheritance. Various distinct steps in transgene uptake by the sperm cell have been described or proposed, including a model based upon endogenous reverse transcriptase activity. Although mature sperm cells are naturally protected against uptake of foreign nucleic acid molecules, certain environmental conditions, for example at key times within the reproductive tract, may reduce this protection, suggesting that SMGT may occasionally take place in nature. If correct, this carries profound implications for evolution and human genetic health. This e-book brings together theoretical and empirical reviews from experts in SMGT, providing comprehensive coverage of the major trends, developments and controversies in this novel field. This e-book is intended as a reference for professional researchers in the field of animal genetic modification (transgenesis) as well as teachers, scientists and physicians interested in medical genetics in general and gene therapy in particular.

http://www.benthamscience.com/ebooks/9781608052370/index.htm

Shemesh, M, Shore L., Stram Y., Harel-Markowitz, E. and Gurevich M. (2012). Manipulation of sperm for efficient production of transgenic calves and chicks. In: Sperm-mediated Gene Transfer: Concepts and Controversies. (ed. K. Smith) pp. 130-145. Bentham E-book

The current method of micromanipulation used for domestic animals results in less than 1% transgenic animals. This makes it extremely difficult to produce transgenic cows and is not feasible for producing transgenic chickens. The purpose of this work was to find a more efficient method for producing transgenic calves and chicks using a combination of two techniques, lipofection and restriction enzyme mediated insertion (REMI). Previously investigators were unable to produce transgenic chickens using lipofection alone. On the other hand, injection of isolated sperm nucleus incubated with restriction enzyme into oocytes has only been shown to be effective in frogs. In this study, we demonstrated for the first time, that lipofection of both DNA and restriction enzyme could be used to successfully integrate DNA into the sperm genome DNA and then used for routine AI to produce transgenic calves and chicks. First it was demonstrated using needle pricking and southern blot analysis of genomic DNA that the restriction enzyme opens up “hot” spots in the sperm genomic DNA. This produces sticky ends by which foreign DNA can be inserted and integrated into the sperm genomic DNA. The “transgenic sperm” thus made were used in IVF and AI to produce embryos expressing a foreign DNA, EGFP (enhanced green fluorescent protein). Using Not I and linearized pEGFP lipofected to sperm for AI resulted with two calves which expressed the exogenous DNA in their lymphocytes as determined by (a) PCR and RT-PCR; (b) specific emission of green fluorescence by the EGFP protein; (c) homology analysis between EGFP DNA and PCR product DNA sequences and (d) Southern blot analysis. Similarly in the chicken, linearized plasmid EGFP sequences with the corresponding restriction enzyme (REMI) were lipofected into the sperm. The transfected sperm were then used for AI in hens and 90% (17/19) of the resultant chicks expressed the exogenous DNA in their lymphocytes as determined by: (a) PCR and RT-PCR; (b) specific emission of green fluorescence by the EGFP; and (c) Southern blot analysis. A complete homology was found between the Jellyfish EGFP DNA and a 313 bp PCR product of DNA from chick blood cells. The procedure was then tested with an additional construct, hFSH. The construct of hFSH consisted of both subunits, α and β and the PCR product used primers for both α and β subunit resulted with a PCR product of 584 bp which was unique to transgenic chickens. The procedure was then used to lipofect a construct of hFSH (Human Follicular Stimulating Hormone) into chicken sperm and used for AI. The resultant offspring were transgenic for at least three generations as determined by: (1) measurement of hFSH protein in chicken blood using enzyme immunoassay and RIA; (2) RT-PCR and PCR; and (3) copy number.

We conclude: (1) that lipofection of both DNA and restriction enzyme into sperm (bovine and chicken) induces the integration of the DNA into the sperm genomic DNA; (2) lipofected sperm can be used in AI to produce a high percentage of transgenic calves and chicks; (3) The integrated gene is expressed in the first, second and third generation; and (4) the method is not limited to specific genes. The technique of lipofection of DNA combined with REMI is therefore an efficient and stable method of producing transgenic domestic animals. Efficient production of transgenic domestic animals could have major impact on gene therapy, improving livestock breeds and the production of valuable pharmaceuticals, e.g. hFSH, which could be extracted from eggs and milk.

Real time RT-PCR for hFSH from second generation transgenic chicken tissues using 1:10 serial dilutions of 0.2 µg/tube of extracted RNA.

The sample amplification curves begin rising between the 35^th and 39^th cycle of PCR, while negative control (not shown) remained horizontal. The differences in the cycle no. of the curves represent differences in the gene expression in different tissues

Environmental estrogens 2012

The Israel Journal of Veterinary Medicine has issued a special issue in honor of Prof. Natan  Ayalon

here


This is the abstract of our contribution.

Effects of Environmental Estrogens on Reproductive Parameters in Domestic Animals

Shemesh, M. and Shore, L.S. Department of Hormone Research, Kimron Veterinary 
Institute, Bet Dagan, POB 12, 50250 Israel.

ABSTRACT

Environmental estrogens are natural products of plant (phytoestrogens) or animal origin (steroidal  estrogens) that have estrogenic properties. They are a major group in a category known as endocrine disruptors. In the field of animal husbandry, the effects of these environmental estrogens are well documented. This paper discusses  the effects of plant estrogens and environmental steroid hormones, such as estrogen and testosterone, as seen in animals in Israel. The areas considered are reproductive disorders, premature udder development, prolapsed oviduct, scrotal atrophy and skewed sex ratios.

Keywords: phytoestrogens, zearalenone, estradiol 17-β, testosterone, testes, oviductal prolapse, scrotum, sex ratio

Israel Journal of Veterinary Medicine  Vol. 67 (1):6-10 March 2012

Effect of estrogen implants on scrotal circumference and tonus in bull calves. Thirteen calves were implanted at 3 mo and their testicles measured at 6 months. Fourteen other calves similarly fed and housed served as controls.  The control circumference (cm) was significantly larger than the treated (P<0.01) and the tonus was significantly lower in treated bulls (P<0.02; 10=very poor; 20=poor; 30=good).

There is scant literature on the effects of exogenous estrogen on bull fertility.  Here is the graph from a short note  that I found.
Infertilité mâle bovine et teneur en coumestrol de la Luzerne.
Infertility in bulls and the concentration of coumestrol in alfalfa.

J.Le Bars, Nurard J.S.  Pharmacologie et Toxicolgie Vétérinaires, INRA Publ. Paris 1982 
Le Colloques de l'I.N.R.A. 8:157-158

 Belgian Blue or Belgian White Blue Bull. Established in the early 20th century, this heavily- (double) muscled, mottled Belgian breed, originally intended for beef and dairy use, is now mainly a beef breed, with bulls usually crossed as terminal sires to produce fast developing veal calves.

(cfgphoto.com copyright, used with permission)

For some reason, the picture of the Belgian Blue is getting a lot of hits.  Here are some more of  7 month old Belgian Blue calves selected for an insemination program taken in 1986 in Belgium (courtesy of Dr. S. Marcus)

Belgium blue calf 01

Belgium blue calf 02

Established in the early 21th century, this heavily-double muscled, mottled breed, originally intended for a scientific experiment. is now a  sporadic superhero.

Cleaning out the drawers and found this little note.

 Pregnancy in a hermaphroditic cow.  High estrogen content of the hermaphroditic testes .

Perl, Smuel; Marcus, Smuel; Shore, Laurence S.; Brenner, Gideon;  Shemesh, Mordechai

A pregnant heifer was examined after it was observed to have masses in the pelvic region.  At slaughter, it was found to have a gravid uterus with a 3 month old fetus.  Testes were observed in the mesovarium .  One of the testes was well formed with an epididymis and seminal vesicular structure. Histological examination of the testis showed Sertoli cells, Leydig cells, germ cells and seminal  vesicles.  The second testis was small and contain only medullary cell mass.  Steroid analysis of the formed testes indicated high levels of estrogen and normal levels of testosterone compared to normal testes [Table 1].

There has been a least one previous report of a pregnant hermaphroditic cow. [Trächtigkeit bein einem Zwitter des Rindes (Hermaphroditaismus verus alternans). Fürst.   Deutsche Tierärztliche Wochenschrift  121 15. April.  59:121, 1952] .   Estrogen has long been known to be present in bull seminal fluid [Payne A, Kelch R, Musich S and Halpern M. Oestrogen content of semen and the effect of exogenous oestradiol-17β on the oestrogen and androgen concentration in semen and blood plasma of bulls. Journal of Reproduction and Fertility  50:17-21 (1977)] and aromatase can be present in all of the various cells of the bovine testes. [Carreau, S.; Hess R.A.  Oestrogens and spermatogenesis Phil. Trans. R. Soc. B 2010 365, 1517-1535.]

Tissue source	Testosterone	Estrogen
	ng/g  fresh wt	ng/g  fresh wt
Hermaphrodite
Interstitial	35.0	2.0
Leydig	8.2	0.3
Epididymal	33.1	0.4
Normal Calf (1 yr)
Interstitial (left)	42.0	<0.1
Interstitial (right)	60.5	<0.1
Epididymal	2.8	<0.1
Epididymal	4.0	<0.1

Estrogen binding receptor 

For many years  the Hormone Research Laboratory made their own cytosolic estrogen receptor.  It was used for the determination of phytoestrogens like coumestrol in alfalfa and zearalenone in corn products.  However, it became  too difficult to  obtain 6 dy old pregnant rabbits so we abandoned the assay.  Efforts to find a replacement have not been successful to date. The most promising system is XDS's Lumi-cell ER based on engineered cells linked to luminiferase.

Seems to work nicely for zearalenone, coumestrol, genistein and diadzein in the expected order.
The upper graph also shows alpha estradiol which usually isn't tested for.  Alpha estradiol is the principal estrogen found in cow blood but is not considered an active estrogen. 

I guess I should mention invitrogen which makes LanthaScreen™ TR-FRET Estrogen Receptor alpha and beta assays.  On paper this sounds impressive.  An assay using recombinant receptor linked to the  rare earth element terbium.  When the receptor is engaged, the terbium shifts its spectrum which can be detected with a spectrophotometer.   They do not offer any training or someplace where you can go to see it in action.  After I ordered it and went over the specs, I saw that it could not possibly work on the spectrophotometer I had even though it stated on their web site that it would work.  After a short email to the company, I was informed that indeed it would not work.  If somebody out there wants the kit, they are welcome to it.  Google states not to put your email on a web site because of spammers so just write me on Facebook or something.  Laurence Shore

Media

http://seattletimes.nwsource.com/html/health/2018947430_spermbank19.html

Originally published August 18, 2012 at 4:41 PM | Page modified August 18, 2012 at 5:20 PM

Israel sperm banks find quality is plummeting
The quality of the product being offered to Israeli sperm banks is falling at an alarming rate, and no one is sure why.

By Edmund Sanders

Los Angeles Times

JERUSALEM — The founder of the Tel Aviv-based specialty firm raves about his product with the same gusto distillers reserve for their top-notch scotch. He's particularly proud of his "premium" line. Sure, it costs a bit more, but it's targeted at a more discriminating client.

Dr. Jacob Ronen is in the sperm business. Among other things, as head of Cryobank Israel, the country's largest private sperm bank, he guarantees that his stable of superior donors includes only tall, twenty-something ex-soldiers whose sperm has passed rigorous genetic testing.

But finding such super sperm isn't as easy as it used to be. Only 1 in 100 donors makes the cut. A decade ago, it was 1 in 10.

It's not just first-rate sperm that's in short supply. All of Israel's half a dozen or so sperm banks are scrambling to keep their liquid-nitrogen freezers stocked.

Simply put, the quality of Israeli sperm is falling at an alarming rate, and no one's sure why.

Fertility is a major issue in Israel, where memories of the Holocaust genocide are fresh, and having children is an entrenched part of Judaism. There's also a political aspect because birthrates among Arabs in Israel have at times been as much as double those of Jews, triggering a population race that some believe could one day affect who controls the land.

So the drop in the quality of sperm is raising some red flags, even though the cause remains a mystery. Theories range from the mundane (carrying cellphones in front pockets) to the far-fetched (depleted uranium from exploded munitions). Some Israeli scientists are looking at naturally occurring hormones, particularly estrogen, in Israel's water and milk. They suggest it's a mark of the country's aggressive dairy-farming methods.

In the microscope

The director of the Hadassah Sperm Bank, Ruth Har-Nir, hunches over a microscope to view a freshly donated specimen and begins to methodically count each squiggly swimmer magnified on the slide.

She is checking the quality of a prospective donor, a young graduate student hoping to earn extra cash. Though sophisticated lab machines could be used to analyze potency, Har-Nir says the old-fashioned method works best.

After a quick scan, she sits up and shakes her head. The number of spermatozoa darting around each tiny grid on the slide is two to four, well below the minimum six required, and nowhere near the 10 to 20 per grid that indicates the concentration the bank likes to see.

Also, rather than surging forward, some of the little guys flit left and right or just stall out, suggesting a weak motility.

"Under no circumstances can we accept sperm of this quality," she says. In the previous three weeks, her bank tested six candidates and rejected all. "This is the trend," she adds.

When Har-Nir helped start the sperm bank in 1991, she says, it turned away about one-third of applicants because of low quality. Using the same standard today, it would reject more than 80 percent. Though the bank relaxed its criteria, it still vetoes about two-thirds.

Long-term change

Har-Nir noticed the problem a decade ago when she began rejecting more and more sperm from otherwise healthy young men. She shared her observations with local fertility doctors, and their research has confirmed her suspicion.

In the past 10 to 15 years, the concentration of sperm samples collected by the bank dropped 37 percent from 106 million cells per milliliter to 67 million, said Dr. Ronit Haimov-Kochman, a leading Israeli infertility researcher at the Hadassah Hebrew University Medical Center.

Though declining sperm quality is an international phenomenon, the change in Israel is occurring at nearly twice the pace as other developed countries, Haimov-Kochman said. If current trends continue, she said, by 2030 the concentration of sperm from Israeli donors will drop below 20 million cells per milliliter, which many international health experts define as abnormal.

There's no evidence declining sperm quality is resulting in fewer babies. The birthrate of Israel's Jewish population has risen in recent decades, thanks largely to an increase in the number of ultraorthodox Jews, who tend to have large families.

Haimov-Kochman estimated infertility rates in Israel have risen from 10 to 15 percent in the past 15 years, but said that's in line with international trends. But she said male infertility — once believed to be the cause about half the time, just as in the U.S. — is now suspected in 70 percent of the Israeli cases.

Most worrisome, she added, is that research has focused on sperm-bank donors, mostly students who are younger and healthier than the general population.

"If this is happening to the guys on our A-team, we might only be seeing the tip of the iceberg," she said.

Banks reaching out

Sperm banks are struggling to cope. Rather than rely on walk-ins as they once did, they use marketing campaigns, posters in college sports centers and Facebook pages to attract virile candidates. The going rate for a donation has doubled in the past 10 years to about $270.

"The decline (in sperm quality) has been dramatic," Cryobank's Ronen said. "It's a shame. We see these macho, beautiful guys come to give donations, but then we're embarrassed to have to tell them that their sperm quality is so low they may actually end up coming back as a client."

He's capitalized on that, though, by offering to freeze sperm of young men with borderline quality who want to set aside a reserve in case their potency declines with age.

Har-Nir said her bank sometimes refers men with the most serious deficiencies for counseling or medical advice. But she emphasizes that rejection by the sperm bank doesn't necessarily mean they won't be able to have children naturally. It just means their sperm isn't, well, commercial quality.

Too much estrogen?

Even with the drop in sperm quality being well-documented, the cause remains unclear and the theories controversial. Some scientists fear Israelis are being overexposed to female hormones.

"People in Israel are getting quite a load of estrogen," said Laurence Shore, a retired hormone and toxicology researcher at the Kimron Veterinary Institute near Tel Aviv. "I don't think it's a good idea to expose children to such high levels of estrogen."

He said no studies have determined estrogen levels in Israel are harming humans, adding that exposure may be too low for that. But he said it might be a factor in the sperm decline.

His research has found Israeli milk and associated products such as butter and cheese can contain 10 times as much estrogen as products from other countries because of Israel's aggressive milk-production practices.

Israel is a world leader in producing milk, pumping twice as much from its cows as other parts of the world, he said. That's partly because cows are milked up to their eighth month of pregnancy, when natural estrogen levels in the milk soar, according to Shore. In nature, he said, cows usually stop giving milk to their own young when they are three months' pregnant with a new calf.

Even though many other nations have adopted similar milking practices, Shore said, Israel is one of the first and most aggressive, so it could be seeing the effect sooner.

Haimov-Kochman is looking into water quality. As a tiny nation with a shortage of water, Israel reclaims much of its used water and sewage, which is processed, used in agriculture and may find its way back into groundwater.

The water, she said, has been found to contain traces of ethinyl estradiol, a synthetic estrogen used in birth-control pills, which gets into the water through the urine of women taking the pills.

"You can't clean this from the water," she said.

Haimov-Kochman is also studying the effect of phthalates, chemicals used in plastic products that are suspected of affecting male reproductive development.

"But I can't prove any of this," she said.

Industry and government scientists dismiss fear about Israel's water and milk as unfounded, saying levels are too small to affect humans.

"Only a tiny part of the total estrogen produced by the cows ends up in the milk," said Dr. Stefan Soback, director of the Ministry of Agriculture's National Residue Control Laboratory. "It is not sufficient to determine estrogen content in milk in order to claim physiological effects to somebody that consumes it."

Batsheva Sobelman of the Los Angeles Times contributed to this report.

Some comments:

"Although estrogenic hormones in milk from nonpregnant cows are in the pg/ml range, milk from pregnant cows can contain 500 ng estradiol/l, 1 µg estrone/l (mostly as conjugated sulfate), and 10 µg progesterone/l [43] (and approximately half of dairy herd is in late pregnancy during milking). Human breast milk for infants contains little estrogen or progesterone since nursing humans are generally not pregnant. Since young children consume about 300–700 ml/day of bovine milk, they may ingest 40 to 100 ng/day of estrogen (estradiol, estrone, and estrone sulfate), and whether this can be considered a safe level is a matter of debate [45]. "

http://old.iupac.org/publications/pac/2003/pdf/7511x1859.pdf

Shore L.S. and Shemesh M. (2003). Naturally produced steroid hormones and their release into the environment. Pure and Applied Chemistry 75:1859–1871

In my opinion, 0.1 microgram per day is not a negligable dose since the daily maintanance level for post-menapausal women is 25 microgram/day. Mice will respond to 50 ng/day of estradiol (A.Kaye, personal observation). I was at the cow meeting a few years ago and some enterprizing young man wanted to extend miliking to 7 and half months. I objected strongly at the time and I trust it was never put into practice. 

Health Effects of Cows’ Milk
http://www.eps1.comlink.ne.jp/~mayus/eng/
Commercial cows’ milk has estrogenic activity as revealed
by the hypertrophic effects on the uteri of young
ovariectomized rats and immature rats
Adapted from:
Ganmaa D, Tezuka H, Enkhmaa D, Hoshi K, Sato A.
Commercial cows’ milk has uterotrophic activity on the uteri of young
ovariectomized rats and immature rats.
International Journal of Cancer 2006;118:2363-65.
Abstract
Cows’ milk has considerable amounts of estrogens, mainly in the form of estrone sulfate. To determine whether the commercial milk has any biologically significant hormonal effects, two series of uterotrophic tests were performed, one with young ovariectomized rats and the other with sexually immature rats. Thirty-six rats were used for each test. They were divided into 3 groups of 12 animals each, and were kept for 7 days on powdered chow with one of three drinking solutions: low-fat milk (experimental), artificial milk (negative control), or artificial milk containing estrone sulfate at 100 ng/ml (positive control). At autopsy, both the wet and blotted uterine weights were measured. The cell heights of uterine epithelia in ovariectomized rats were also determined. In each test, the weights of the uteri in the Low-Fat Milk group were significantly greater than those of the respective weights in the Artificial Milk group (p<0.01). Furthermore, in ovariectomized rats, the uterine epithelial-cell height in the Low-Fat Milk group was significantly greater than that observed in the Artificial Milk group (p<0.01). The uterotrophic effect of 100 ng/ml Estrone Sulfate solution was greater than that of Low-Fat Milk in immature rats (p<0.01), whereas the effect of the solution was almost comparable to that of Low-Fat Milk (p>0.05). In conclusion, commercially available low-fat milk has uterotrophic effects in both young ovariectomized rats and sexually immature rats.

 The standard in US and England is that cows are milked till 220 days or 60 dry days before calving.  Cows are pregnant the same amount of time as humans (283 days and have the same development rate, i.e. differentiation of the sexual organs occurs between 45-90 days of gestation.)