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What are Probiotics?

There are billions of bacteria living in the digestive system of every human, many of which are "friendly" bacteria that contribute to the health and balance of the intestinal tract. This normal microflora prevents the overgrowth of potentially pathogenic bacteria in the intestinal tract.

Sometimes, due to viral or bacterial infections, diet, lifestyle changes, antibiotic use, and other factors, the normal bacteria in our intestinal tract become depleted, allowing the potentially pathogenic bacteria to overgrow. The result may be diarrhea, gas, or other gastrointestinal symptoms.

Nutritional supplements with probiotics contain the "friendly" bacteria that your body needs. A diet including probiotics may have a beneficial effect on the intestinal tract and help maintain a healthy digestive system.

Probiotic foods are also known as functional foods, which have been in our diets for years. A functional food is a product that is consumed every day and has been shown through clinical studies to maintain human health or to promote human well-being better than similar traditional foods.

 

What to look for in a probiotic

 

                      Probiotics short course and strains compiled from several sources.

 

                      1. Trillions of microorganisms live on and inside us. Most are good, some are harmful. The beneficial ones are called "probiotic microorganisms." They improve the environment of the intestinal tract.The use of good probiotics is important in healing many chronic gastrointestinal problems that are so often              associated in those with ASD. Some experts feel that ASD children need several times the amount of probiotics than those without GI problems, due to the frequency of dysbiosis (overgrowth of yeast, bacteria, etc.) and "Leaky Gut"

                      (intestinal permeability problems). 

                      2 Scientific studies over the last 50 years show that probiotic organisms can improve the nutritional quality of foods, produce antibiotics, anticarcinogens, and substances that break down and recycle toxins for their human host.  

                      3. The major benefits of adding probiotic organisms to the diet: boosts immune system, inhibits disease causing organisms, improved digestion, vitamin synthesis, detoxification and protection from toxins, prevents diarrhea from various causes, reduced risk of irritable bowel syndromes, cancer-protective            effects, increased nutrient absorption, improves resistance to allergies, reduces yeast and other infections.

 

                      4. Lactobacillus acidophilus bacteria reside mostly in the small intestine, and Bifidobacterium bifidum are found in the large intestine (colon). Whether taking a mixed-species product or a single species is better has not been determined. There are mixed opinions. Some will decide to choose a single strain of friendly flora because of the proven effectiveness of that particular                  probiotic strain. Taking this strain for its specific properties can be very helpful. However, over an extended period of time, you may want to include a variety of strains.  

                      5. Capsules are the preferred way to take probiotics because there is more protection from contamination, oxygen, and moisture, and capsules maintain organism integrity. Dairy products that contain added organisms like lactobacillus provide a mild dose of probiotics, if you can have it. 

                      6. Generally higher more therapeutic doses of probiotics are need when first addressing GI symptoms. Probiotic strength is measured in CFUs (colony forming units) per capsule. You may want to take one or several daily. For therapeutic                    benefits, references varied widely from 250 million – 20 billion viable

organisms/day. It is best to check with your doctor, or you can start slowly and build-up to a level you feel is most beneficial. 

                      7. Talk to others and about quality issues, and what works best. Choose a probiotic that has been extensively researched with a great deal of scientific support behind it. Don't buy any product unless it has the manufacture date right on the bottle. Probiotic products, especially lactobacillus and                    bifidobacterium, lose a lot of potency after 4-10 months. Products last longer when they are refrigerated, although some product do not require refrigeration.

 

                      8. When to take probiotics varies by brand. Always check the label and follow the recommendation of the manufacturer. Some say to take on an empty stomach; some say with food so the food can buffer the organisms; some say in the morning because of stomach acid content; some can be taken anytime. The acid           and salts in the gut will harm certain probiotics. Manufacturers take this into

account when designing a formulation and preparing the capsules. Some capsules are specially coated so the microorganisms will safely reach their destination.

                      Others need to be taken at certain times for optimum performance for that product. Probiotics are not adversely affected by the use of enzymes. They can both be taken during at mealtime.

 

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                      Probiotic Strains

 

                      This is a list of most strains of probiotics used. The * indicates the most prevalent ones in products. You can do a search by Probiotics or the species you are in terested in to find out more.

 

                      Lactobacillus acidophilus *

                      Lactobacillus brevis

                      Lactobacillus bulgaricus

                      Lactobacillus casei subspecies casei

                      Lactobacillus casei, subspecies paracasei

                      Lactobacillus casei, subspecies rhamnosus *

                      Lactobacillus fermentum

                      Lactobacillus rhamnosus (casei) *

                      Lactobacillus caucasicus

                      Lactobacillus helveticus

                      Lactobacillus lactis

                      Lactobacillus plantarum

                      Lactobacillus reuteri

 

                      Bifidobacterium bifidum (lactis) *

                      Bifidobacterium breve subspecies breve

                      Bifidobacterium infantis

                      Bifidobacterium licheniformis

                      Bifidobacterium longum *

                      Bifidobacterium subtilus

 

                      Enterococcus faecium

                      Fructooligosaccharides (scFOS) -feeds the friendly bacteria

                      Streptococcus cremoris

                      Streptococcus faecium

                      Streptococcus infantis

                      Streptococcus thermophilus

 

                      There is substantial research done on one probiotic in particular called Culturelle GG

                      Culturelle GG studies

 

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                      Probiotic Descriptions

 

                      Thanks to John Hedy of http://www.vitallifevitamins.com/

                      for the following information.

 

                      1. Bifidobacterium Bifidum

 

                      B. bifidum is a prominent probiotic microorganism that takes up residence primarily in the mucous membrane lining of the large intestines and the vaginal tract. B. bifidum prevents the colonization of invading pathogenic bacteria by attaching to the intestinal wall, crowding out and taking nutrients from these unfriendly bacteria and yeast. B. bifidum produces lactic and      acetic acids, which lower the intestinal pH and further inhibit the undesirable bacteria from growing. Research on Bifidobacteria has established that these organisms enhance the assimilation of minerals such as iron, calcium, magnesium and zinc.

 

 

                      2. Bifidobacterium Infantis

 

                      Bifidobacterium infantis is an important organism shown to stimulate production of immunomodulating agents such as cytokines.

                      Bacteriocidal activity is also observed against such pathogens as  Clostridia, Salmonella, and Shigella. 

 

                      3. Bifidobacterium Longum

 

                      Bifidobacterium longum is a very abundant organism found in the large intestine. It plays a role in preventing the colonization of invading pathogenic bacteria by attaching to the intestinal wall and crowding out unfriendly bacteria and yeast. Along with other microorganisms, it produces lactic and acetic acids that lower the intestinal pH and further inhibit the undesirable bacteria. B. longum has, in clinical studies, been found to reduce the frequency of gastrointestinal disorders (diarrhea, nausea, etc.) during antibiotic use. 

 

                      4. Enterococcus Faecium

 

                      E. faecium has been shown to be important in the nutritional support of diarrheal diseases, especially in cases where pathogenic microbes, such as rotavirus, invade the bowel. This particular organism only transiently colonizes the GI tract. A recent study indicated that an

                      E. faecium-containing yogurt was able to significantly lower LDL cholesterol. E. faecium is safe, and has been researched extensively by the World Health Organization. 

 

                      5. Fructooligosaccharides & Inulin

 

                      FOS and inulin are non-digestible oligosaccharides that help promote the growth and activity of friendly bacteria in the intestinal tract.

                      These oligosaccharides are non-caloric compounds that can not be broken down by our digestive enzymes and therefore do not adversely affect blood sugar levels. Research has shown that both FOS and insulin enhance the growth of lactic bacteria, especially

Bifidobacteria, and inhibit the growth of a variety of undesirable

organisms.

 

                      6. Lactobacillus Acidophilus

 

                      L. acidophilus is one of the most important microorganisms found in the small intestines. It is known to implant itself on the intestinal wall, and in the lining of the wall of the vagina, cervix, and urethra. It performs many critical functions including inhibiting pathogenic organisms and preventing them from multiplying and colonizing.

 

                      It is well documented that L. acidophilus produces natural antibiotics like lactocidin, acidophilin, etc., which enhances

resistance or immunity. L. acidophilus has known antimicrobial

activity against Staphylococcus aureus, Salmonella, E.coli and

Candida albicans.

 

                      7. Lactobacillus Brevis

 

                      Lactobacillus brevis is a lactic acid producing organism important in the synthesis of vitamins D and K.

 

                      8. Lactobacillus Bulgaricus

 

                      Lactobacillus bulgaricus is considered a transient microorganism that does not implant in the intestinal tract, but still provides an important protective role. This organism is used extensively in the commercial fermentation of yogurt. Production of lactic acid by the bacterium provides a favorable environment for the growth of other lactobacilli and bifidobacteria residing in the intestine. Studies indicate that certain strains of L. bulgaricus stimulate production  of interferon and tumor necrosis factor, thus establishing a potential role in modulating the immune system.

 

 

                      9. Lactobacillus Casei

 

                      Lactobacillus casei is closely related to the L. rhamnosus and L. acidophilus strains with some of the same immuno-modulating effects as other Lactobacilli. L. casei has several health-promoting effects provided through the production of bacteriocins, compounds that inhibit the growth of pathogenic bacteria in the small intestine.

 

 

                      10. Lactobacillus Plantarum

 

                      Lactobacillus plantarum secretes the naturally occurring antibiotic lactolin, and is also known to have the ability to synthesize the amino acid L-lysine, which has beneficial anti-viral activities. L. plantarum also produces glycolytic enzymes shown to degrade cyanogenic glycosides and is effective in eliminating nitrate while  producing nitric oxide. This probiotic can preserve key nutrients,vitamins, and antioxidants, eliminate toxic components from food, and  eradicate pathogens such as S. aureus from fermented food. L. plantarum-fermented oat given to healthy volunteers significantly reduced a number of potential pathogens in the gut.

 

 

                      11. Lactobacillus Rhamnosus

 

                      Lactobacillus rhamnosus is primarily found in the small bowel and vaginal tract and is beneficial in inhibiting those bacteria involved in vaginal and urinary tract infections. L. rhamnosus is very prolific in growth, has a high tolerance (resistance) to bile salts, adheres to the intestinal mucosa, and protects the intestinal tract against the invasion of harmful microorganisms. Additionally, this  organism favorably affects lactose intolerance. A recent double-blinded, placebo-controlled study suggests that this probiotic bacteria may down-regulate hypersensitivity reactions and intestinal inflammation in patients with atopic eczema and food allergies. L.rhamnosus has been found to have significant benefits in the nutrition and well-being of infants and in the elderly. According to research with this strain, administration of L. rhamnosus is most  helpful in inhibiting early intestinal infections in infants. This species of Lactobacillus does not only colonize, acidify and protect the small intestine, but it can quickly establish itself in the large intestine, inhibit the growth of streptococci and clostridia, create anaerobic conditions which favor the implantation of bifidobacteria,and produce biologically desirable lactic acid.

 

                      12. Lactobacillus Salivarius

 

                      Lactobacillus salivarius is important in normalizing the gut flora of those dealing with chronic bowel conditions and shows potential as an effective inhibitor of H. pylori, an organism associated with the occurrence of ulcers.

 

 

                      13. Streptococcus Thermophilus

 

                      Streptococcus thermophilus, in combination with L. bulgaricus, is used commercially to produce yogurt. This organism is known to be efficient in breaking down lactose by producing the enzyme lactase.

                      Those who are lactose-intolerant may be greatly helped by supplementation with this particular strain. Cytokine production is stimulated in tissue cultured cells by this bacterium.

 

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                      Combinations

 

                      1. L. acidophilus, L. rhamnosus

                      Many researchers now believe the myriad health benefits of L. acidophilus are also attributable to L. rhamnosus. These two species are perhaps the most important Lactobacilli in the small intestine.

 

 

                      2. E. faecium

                      This probiotic has become so popular with health professionals over the years because of the proven therapeutic value of E. faecium. This species shows strong activity against a variety of pathogenic organisms. In several studies it has proven resistant to a wide variety of antibiotics and, in one  study, proved more effective than L. acidophilus in shortening the duration of diarrheal episodes. E. faecium is a natural resident of the human intestinal tract.

 

 

                      3. L. rhamnosous, L. acidophilus, B. lactis, Streptococcus

                      thermophilus, L. bulgaricus

                      These are five extensively researched strains of friendly bacteria. These strains maintain viability in acidic environs as may be found in the stomach, and are tolerant to compounds found in the intestine such as bile. Clinical research has documented the usefulness of these probiotic strains as an adjunct to the management of gastrointestinal disorders, including:                     Antibiotic-associated decrease in friendly bacteria, Prevention of

Clostridial colonization, Traveler's diarrhea, Diarrhea associated

with rotaviral gastroenteritis, Acute non specific diarrhea,

Constipation, Enhancement of immune response to rotaviral infection

and adjuvant to rotavirus vaccine, Alleviation of intestinal

inflammation and permeability, Amelioration of food allergies,

especially lactose intolerance, Enhancement of the intestine's

immunological barrier function, Intestinal production of short-chain

fatty acids.

 

 

                      4. L. casei, L. rhamnosus, L. acidophilus, B. longum

 

                      Important studies have demonstrated the immune-enhancing properties of lactic acid bacteria. L. casei has been named in a significant number of these studies. L. rhamnosus, until recently, was subsumed under the heading of L. casei and likely possesses similar immune-potentiating characteristics as L. casei. A strain of L. rhamnosus was also recently shown to mitigate the effects of food allergy on infants with atopic dermatitis -- a reduction in

intestinal inflammation was considered a key factor in bringing about the improvements observed. Numerous studies have shown probiotic organisms to be effective in reducing lactose intolerance, relieving constipation, preventing gastrointestinal infections, enhancing immune activity and, in some studies, reducing cholesterol.

 

 

                      5. Culturelle Lactobacillus GG: Lactobacillus GG.

 

                      Culturelle is the only probiotic supplement containing Lactobacillus GG.

                      In 1985, Drs. Sherwood Gorbach and Barry Golden isolated a new strain of Lactobacillus that appears to be ideal for use in humans. The strain, named Lactobacillus GG (after the surnames of its inventors), is resistant to   stomach acid and bile, allowing it to survive its passage through the digestive tract and reach the large intestine intact. Once there it shows an exceptional ability to adhere to the intestinal mucosa and proliferate.

 

                      see Research on Culturelle

 

                      Will enzymes interfere with probiotics/probiotic foods? 

                      The enzymes in question are usually proteases. Probiotics consist mainly of proteins so there is the thinking that protease enzymes might  break down the probiotics or make it harder for them to securely attach and anchor in gut. I searched a lot of the research literature several  times on this.

 

                      It depends greatly on the probiotic strain in question and how it is manufacturered. Some strains are totally unaffected by enzymes, whereas  other strains are slightly affected, and others very affected. Some probiotics are destined to function in the small intestine and some in the large.

 

                      The other consideration is how it is manufacturered. Some are enterically coated, some are not. Some can be mixed with foods, some can not. Some *must* be taken with meals, some *must* be taken between meals. Some in the morning, some it doesn't matter. 

                      If you aren't sure, the safest bet is to just give the probiotic at the end of the meal (the enzymes are given at the beginning) or between meals (unless it specifies with food). I always gave probiotics at bedtime. 

                      Check the label of the probiotic and call the manufacturer of the probiotic to be sure how *their* product needs to be taken. They could comment on their own products. You can also just try giving probiotic with enzymes, and then without...and see if it makes any noticeable change. 

                      Should enzymes be taken with probiotics or probiotic foods, such as the young green coconut kefir, cultured vegetables and nut yogurts recommended on BED and SCD diets? 

                      This is generally fine. I hope to get more definitive information on this soon. The difference is that the probiotics in food are in a whole-food form. Probiotics 'grown' or derived in food are producing digestive enzymes anyway. So they are already there. In fact, this is one of the benefits that yogurt and other fermented foods supply: digestive enzymes to your body! 

                      The probiotics in food are already 'established' and hard at work digesting the food as you are eating it. Probiotics in supplements are not.

                      They are preserved in the capsule or tablet. They get to the gut, then they need to 'attach' and colonize. Then they start digesting food as it comes down the hatch. 

                      Some of the supplement makers have the sales line: Our probiotic supplements provide much higer counts of bacteria than a cup of yogurt.

                      You would need to eat 6 cups of yogurt to equal it. However, many of the probiotics in capsule may very well get wiped out going through the gut and attempting to colonize. 

                      The yogurt makers will say: You don't need that high a culture count because the probiotics are already established and going in a whole-food form. So in the final tally, the yogurt culture may well outperform the supplement. In addition, the yogurt supplies other beneficial factors that a supplement does not.

                     

Probiotic Products
Also see:
Gastrointestinal Support and Microbial Balancers

 

The gastrointestinal (GI tract) is actually a complex micro-ecosystem in which the mucosal lining of the host coexists with billions of microorganisms that live on or attached to the lining. These "probiotic" bacteria are provided with shelter and support, and copious amounts of food substrates. The body benefits from the vitamins and other useful metabolic products these bacteria produce. Other, less beneficial micro-organisms are also present and compete with the probiotics. Dietary supplementation with potent probiotic cultures assists the host in maintaining a healthy probiotic balance. 

 

Allergy Research Group's probiotic products are non-dairy and are generally well-tolerated. They should be kept away from extremes of heat or cold. Their potency is best maintained by storing them refrigerated (but not frozen) until just before opening, then keeping the opened bottle in a cool, dry place and using up the contents within 4-6 weeks.  These formulations take advantage of the powers of certain plant extracts to favorably affect the intestinal balance of micro-organisms.

* This statement has not been evaluated by the U.S. Food and Drug Administration. The product is not intended to diagnose, treat, cure, or prevent any disease.

 


BifidoLife with L. sporogenes 60 capsules
# 51910
Click here to order

Bifidobacteria is the largest probiotics group in the large intestines.  They are comparatively delicate, however, and their numbers can be depleted by toxins in the intestines or by other stressors.  Lactobacillus sporogenes (Bacillus coagulans) is a non-dairy probiotic strain uniquely "pre-encapsulated" in spores that resist the action of antibiotics and gastric juices.

Each capsule contains:
Lactobacillus rhamnosus 2.2 Billion
Bifodobacterium breve 1.2 Billion
Bifodobacterium longum 1.2 Billion
Lactobacillus sporogenes 360 Million
Lactobacillus acidophilus 240 Million

Suggested Use:
As a dietary supplement, 1 capsule two to three times daily, or as directed by a healthcare practitioner.

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Gastro Flora 60 capsules
# 52460
Click here to order

This combination of 4 beneficial ("probiotic") bacteria is designed to support optimal equilibrium of the flora of the GI tract.  The 4 probiotic bacteria consist of Lactobacillus acidophilus, L. rhamnosus, L. casei, and Bifidobacterium longum.  Free of milk, grains, yeast, soy, and other common allergens.

Each capsule contains:
Lactobacillus casei 1.6 Billion
Lactobacillus rhamnosus 1.6 Billion
Lactobacillus acidophilus 0.4 Billion
Bifodobacterium longum 0.4 Billion

Suggested Use:
As a dietary supplement, 1 to 3 capsules two or three times daily, or as directed by a healthcare practitioner.  To maintain potency, store tightly closed and refrigerated.  Short-term heat exposure is acceptable.

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Lactobacillus GG (Culturelle®)
# 73390
Click here to order

Lactobacillus GG is an extensively studied lactose and dairy free strain of friendly intestinal bacteria, naturally occurring in the digestive tract.  Together with Lactobacillus rhamnosus LGG is a subspecies of Lactobacillus casei.  It was discovered and patented by researchers Drs. Gorbach and Goldin at Tufts University.  LGG is a member of the L. acidophilus family of friendly probiotic bacteria.  To date it is the best-studied and most extensively documented probiotic lactic acid bacteria strain in the world, containing 20 billion viable organisms per capsule.

Each capsule contains:
Lactobacillus GG 40 mg (20 Billion)

Suggested Use:
As a dietary supplement, take 1 capsule per day, preferably on an empty stomach, or as directed by a healthcare practitioner.

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LactoBlend 100 capsules
# 52780
Click here to order

Contains three particularly hardy strains of the friendly probiotic bacteria lactobacillus.  This may be a more appropriate product for individuals who do not derive benefit from less hardy strains of lactobacillus such as acidophilus.

Each capsule contains:
L. plantarum 10 Billion
L. salivarius 4 Billion
L. rhamnosus 3 Billion

Suggested Use:
As a dietary supplement, 1 capsule one to three times per day, on an empty stomach, sensitive individuals may want to take with food, or as directed by a healthcare practitioner.

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SymBiotics with FOS 140 grams
# 51130
Click here to order

This product puts the probiotics all together!  Some suppliers claim that the probiotic strains can only work if given one at a time, and that certain single strains have near magical properties.  Yet the intestinal micro-ecosystem typically carries up to 400 strains of bacteria.  From the point of view of intestinal health, a product that provides bacteria for the different probiotic "niches" makes a lot more sense.  The nine strains in SymBiotics include Lactobacillus acidophilus; L. rhamnosus A and L. rhamnosus B (both more versatile and hardier than L. acidophilus); L. bulgaricus and L. casei (major contributors to fermented foods); and L. sporogenes (Bacillus coagulans), a particularly hardy and antibiotic resistant probiotic strain.*  Bifidobacterium longum and B. breve, two of the hardier bifidobacteria , are also present in high potency, as is Streptococcus thermophilus.  The bifidobacteria thrive on the FOS provided in this formulation, and ascorbic acid (vitamin C) is added as a stabilizer.  SymBiotics with FOS contains no dairy material and provides more than 4 billion viable organisms per serving.

Each capsule contains:
Calories 10
Total Carbohydrates 3 g
   Sugars 3 g
Vitamin C (as Ascorbic Acid) 180 mg
Fructooligosaccharides 3 g
Lactobacilli 3 Billion
Bifidobacteria 0.8 Billion
Streptococcus thermophilus 0.4 Billion

Suggested Use:
As a dietary supplement, 1 level teaspoon two or three times daily, or as directed by a healthcare practitioner.  To maintain potency, store tightly closed and refrigerated.  Short-term heat exposure is acceptable.

CAUTION: Some individuals using FOS experience gastrointestinal bloating that does not subside after 1 of 2 weeks of use.  In those cases, FOS should be discontinued.  Recent clinical experience by some practitioners suggests that there are some unfriendly microbial invaders that are suspected of feeding on FOS.  Symptoms of that occurring would be as indicated above.

*These statements has not been evaluated by the U.S. Food and Drug Administration.  The product is not intended to diagnose, treat, cure, or prevent any disease.

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The bifidobacteria group are the most common probiotics in the large intestine. They are compartively delicate, however, and their numbers can be depleted by toxins in the intestines or by other stressors. Lactobacillus sporogenes (Bacillus coagulans) is a probiotic strain uniquely "preencapsulated" in spores that resist the action of antibiotics and gastric juices; it is non-dairy and is cultured on malt.

Each capsule contains a minimum of:

Bifidobacteria..........2.4 billion viable cells

Lactobacillus sporogenes..........360 million viable cells (Bacillus coagulants)

Available in bottles of 60 capsules. Non-dairy.

Suggested Use:

As a dietary supplement, one (1) capsule two or three times per day with meals, or as directed by a health practitioner. TO MAINTAIN POTENCY, STORE REFRIGERATED UNTIL JUST BEFORE OPENING. DO NOT FREEZE. SHORT TERM HEAT EXPOSURE IS ACCEPTABLE.

This statement has not been evaluated by the (FDA). These products are not intended to diagnose, treat, cure, or prevent any disease.


Availability: Usually ships in 2-3 business days.

ZEE-TOX PLUS

(ACIDIFIED ZEOLITES AND PROBIOTICS)

 COMPOSITION:
SiO2 - 64.2% 
Fe2O3 - 3.4%
Mgo - 2.6%
K2O - 0.4%
P - 015%
AI2O3 - 19.45% 
CaO - 5.6%
Na2O - 4.1 %
Mn - 0.03%

ORGANIC ACIDS
Acetic Acid.
Citric Acid 
Propionic Acid.

PROBIOTICS :
Lactobacillus sporogenes - 5 x 1010
Saccharomyces Cerevisiae - 2 x 1O12
Bacillus Coagulans - 5 x 1010
Streptococcus faecium - 5 x 106

ZEE-TOX PLUS.

·         Prevents damage due to Mycotoxins like Aflatoxin Vomitoxin, Ocharatoxin and Zearalenone.

·         Inhibits mold growth

·         Acts as selective toxin binder.

·         Improves flow property of feed.

·         Enhances biosynthesis of digestive enzymes.

·         Probiotics result in lactate production thereby establishing healthy gut flora.

·         Competitive exclusion of enterolnvasive and epterotoxic bacterial pathogens from the gut of birds.


 DOSAGE:

Moisture Dose
Upto 15% 1.0Kg/ton of feed
Above 15% 1.5kg/ton of feed

PRESENTATION: Zee-Tox plus is available in 20kg Poly pack.

Lactobacillus sporogenes is a harmless type of bacterium naturally occurring in the intestine and constitutes a major part of intestinal flora. Lactobacillus sporogenes plays a very important role in upkeep of health and is responsible for the synthesis of B-complex factors,Vitamin K, digestive enzymes required in digestion of various carbohydrate, fat and protein foods and their absorption into body.

Lactobacillus sporogenes has many interesting properties, the most notable of which is its ability to transform into small oval bodies known as spores.

The spores are extremely stable, have a long life and are resistant to high temperature,gastric acid and bile acid.

In the host these spores survive gastric acidity, germinate in the duodenum, proliferate in the small intestine, produce abundant lactic acid, prevent proliferation of harmful putrefactive bacteria and create an environment for normal conditioning of the gastrointestinal tract.

Lactobacillus sporogenes does not consume vitamins during its proliferation or growth. It also produces biologically active L (+) Lactic acid, which is completely metabolized leading to glycogen synthesis and ideally fulfills WHO recommendation for products of infant nutrition programs. Currently Lactobacillus sporogenes formulations are used for various indications like diarrhea (adult, infant and neonatal) lactose intolerance, candidiosis, dyspepsia,constipation, colities etc.

In fact beyond its well-established usefulness in promotiong digestive health, its most impressive research finding is in the area of containing CHOLESTEROL LEVELS. Lactobacillus sporogenes has been documented to significantly lower total serum cholesterol & the LDL-cholesterol to HDL-cholesterol ratio without any side effect in hyper cholesteroaemea.

LACTOBACILLUS spores are capable of providing protection from pathogenic invasions into intestinal tract and help to restore the normal balance of the intestinal flora after antimicrobial drugs treatment, whereas the non-sporeforming (vegetative cells) are a total failure. Thus they possess ideal and desired characteristics of a drug that does not cause any pathological or pharmacological side effects. PROBIOTIC LACTOBACILLUS SPOROGENES The natural alternative for animal health care.

THERAPEUTIC USES

COMPATIBILITY

ACUTE ENTERITIS

POWDER / TABLETS

APTHOUS STOMATITIS

B-COMPLEX VITAMINS,MULTI -VITAMINS

CANDIDIOSIS

DIGESTIVE ENZYMES + ELETROLYTES

CONSTIPATION

SULPHAMETHAXAZOLE AND / OR TMP

COLITIS

METRONIDAZOLE / TINIDAZOLE

CHOLESTEROL DEGRADATION

ANTIDIARRHEALS

DIARRHEA/NEONATAL DIARRHEA

ANTIBIOTICS LIKE TETRACYCLINE/ CHLORAMPHENICOL / AMPICILLIN / AMOXYCILLIN

DYSPEPSIA

 

LACTOSE INTOLERANCE

 

RECONDITIONING OF G.I TRACT AFTER ANTIBIOTIC THERAPY

PROTEINS,CARBOHYDRATES AND MINERALS.

 

PROBIOTIC LACTOBACILLUS SPOROGENES
( The natural alternative )

 

  • LIMITS THE ACTIVITY OF HARMFUL PATHOGENIC BACTERIA ( E.COLI, SALMONELLA, CAMPYLOBACTER ETC)

 

  • MAINTAINS A HEALTHY, BALANCED GUT MICROFLORA

 

  • IMPROVES PERFORMANCE

 

  • REDUCES DIGESTIVE UPSETS

 

  • IMPORVES FCR ( FEED CONVERSION RATIO)

 

  • IMPROVES RESISTANCE TO STRESS AND DISEASE

 

  • LOWERS MORTALITY - IMPROVES PROFITABILITY

 

Table. 1. Percentage of top dairies using various feed ingredients.

Ingredient

Percent

Sodium bicarbonate

78.8

Yeast

58.3

Magnesium oxide

54.6

Megalac1

46.2

Zinc methionine2

42.4

Niacin

42.2

Anionic salts

33.3

4-Plex2

31.1

Biotin

1.5

Probiotics

0.8

1Church and Dwight Co., Inc., Princeton, NJ
2Zinpro Corporation, Eden Prairie, MN
Kellogg, D. W., et al. 2000

       

TITLE EFFECT OF A PROBIOTIC YEAST SUPPLEMENT ON PERFORMANCE OF TRANSITION HOLSTEIN COWS
 

AUTHORS     D.R. McKnight, P.H. Sharpe and N.K. Gurung
                        Kemptville Campus, University of Guelph
                        Kemptville, Ontario K0G 1J0
 

Effect of a probiotic yeast supplement on performance of transition holstein cows. D. R. McKnight, P.H. Sharpe, and N.K.Gurung. Kemptville College, University of Guelph, Kemptville, On, KOG IJO.
 

Twenty-eight Holstein cows and heifers were randomly assigned in pairs by parity and date of calving to two treatments. Control cows were supplemented with 30 g/h /d of a placebo and treated cows with 30 g/h/d of Fastrack L.S. 4000 to determine the effects of feeding Fastrack® yeast probiotic culture on dry matter intake, milk yield and composition. Fastrack® was fed from approximately 21 days prepartum to exactly 15 weeks postpartum. The prepartum diet consisted of a mixture of haylage and corn silage (50:50 DMB), free-choice grass hay and lead feeding of milking cow TMR starting 2 weeks prepartum. The postpartum diet consisted of a TMR based on corn silage, haylage, high moisture corn and pelleted supplement. The Fastrack® treatment was topdressed once daily at 9:30 a.m. at the first daily feeding. Cows were housed in a two row tie-stall barn and fed free choice three times daily. Daily intake and weighbacks were recorded beginning 21 days prior to expected calving through weeks 15 of lactation. Body weight, body condition score, milk yield and milk composition were recorded weekly. Least squares means for intake, milk yield and milk composition were unaffected by treatment. Overall dry matter intake and regain of body weight and condition score was lower than anticipated. The Fastrack® yeast probiotic culture fed at 30g/h/d to dry and lactating cows had no effects on prepartum or postpartum dry matter intake or milk yield and composition.

Key words: Dairy, probiotic, yeast, transition
 

2. INTRODUCTION
 

In early lactation, high producing dairy cows often fail to consume sufficient feed and generally are in negative energy balance. Increasing the proportion of concentrates above 55-60% of total ration dry matter (DM) to provide higher energy density may result in problems such as low appetite, rumen acidosis, reduced digestibility, milk fat depression and possibly higher incidences of displaced abomasums, milk fever, ketosis and retained placentas (Clark and Davies, 1980). Bertics et al. (1992) and Van Saun et al. (1993) have demonstrated depression of dry matter intake (DMI) of approximately 40% beginning at 7 to 10 days prepartum.
 

Any factor that would improve prepartum DMI, or increase the rate of increase of DMI postpartum, should be beneficial in increased milk production and/or reduced metabolic problems. Robinson (1997) assessed the effect of a yeast culture fed to cows immediately pre and postpartum. Cows supplemented with yeast culture lost less body condition prepartum, but there were no differences in pre or postpartum DMI. However, Wohlt et al. (1998) reported significantly improved dry matter intake and milk yield (P<0.05).
 

Probiotics are a rapidly growing area of dairy nutrition. Several commercial products are available on the market and are being widely promoted to dairy farmers as a means of getting fresh cows on feed quicker and reducing metabolic problems. One such product is Fastrack® LS-4000 probiotic pack (Conklin Co. Inc., Minnesota, USA) which contains probiotics and yeast. Probiotics are defined as, "a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance" (Fuller, 1989). The "Fastrack® L.S.-4000" contains lactobacillus acidophilus and Streptococcus faecium, yeast, and fermentation products. McGilliard and Stallings (1998) reported that a microbial and enzyme supplement increased milk yields in 31 herds and decreased the milk yield of 15 herds. Jaquette et al.(1998) and Ware et al.(1998) reported significantly increased milk production from feeding a lactobacillus acidophilus supplement. In contrast Soder and Holden (1999) in a controlled study, reported no effect on intake or milk yield from feeding yeast cultures and enzymes.
 

The objective of this study was to examine the impact of a probiotic/yeast supplement to the diet for approximately 21 days prepartum and exactly 15 weeks postpartum on performance of dairy cows.
 

3. MATERIALS AND METHODS

Cows and heifers were blocked into pairs, based on lactation number on day 21 prepartum. Two treatment groups were established. Control was supplemented with 30g/head/day of a sugar placebo, while treatment animals received 30g/head/day of Fastrack® L.S.-4000. Supplement was topdressed in the 9:30 am feeding starting approximately 21 days prepartum.
 

Cows and heifers were tethered in tie-stalls and given free access to water. Each animal began the experiment at 21 days prior to the expected calving date. Mature cows and heifers received 20 kg and 15 kg respectively of haylage/corn silage (50:50 dry matter basis), free choice grass hay and 100 grams/head/day of Co-Op HMG. XP Dairy Mineral Premix. At 14 days prepartum lead feeding began. Cows and heifers were gradually increased in total mixed ration (TMR) and decreased in haylage/corn silage until cows and heifers were receiving 15 kg and 10 kg of TMR respectively. The TMR was formulated to support 45 litres of milk production. Free choice grass hay and mineral supplement continued. One week prepartum cows and heifers were moved into maternity pens for calving.
 

After calving, cows were re-tethered and increased in TMR and reduced in hay as rapidly as possible. Cows continued to receive the control and treatment supplement for 15 weeks postpartum as described above. TMR was be fed 3 times daily at 9:30 am, 3:00 ppm and 9:00 p.m.
 

The ingredient and composition of the TMR, Grain Mix Supplement and Topdress are shown in Tables 1 - 3. Corn silage and haylage were tested every 2 weeks for dry matter content and the TMR adjusted accordingly. TMR was sampled weekly and composited by month. Each batch of mixed concentrates was sampled upon delivery and composited to create one composite sample for each of the two mixed concentrates. Orts were sampled on a day 7 of each week and composited by cow to create a single prepartum composite sample of orts and a single postpartum composite sample of orts.

All feedstuffs were analyzed by Agri-Food Laboratories in Guelph, Ontario. Milk samples were analyzed through Ontario Dairy Herd Improvement Milk Recording Services. Cows were milked daily at 6:00 am and 4:00 p.m. Milk volume and composited (am and pm) samples for compositional analysis were collected weekly. All experimental cows were weighed and assessed for body condition score (BCS) weekly. Feed was fed for ad libitum intake (5 to l0% orts). All animal use was according to standards approved by the Canadian Council of Animal Care.
 

Weekly cow data was analyzed by ANOVA for repeated measures (Gill, 1986) using general linear models procedure (SAS, 1985). All statements of significance were based on the 0.05 level of probability.
 

The composition of the TMR used in the experiment is presented in Table 1. The trial ran from June/98 until August/99. Over that period of time 14 cows completed data collection on each treatment. Calving dates were unpredictable resulting in some cows receiving Fastrack for 3-4 weeks prepartum and some receiving very little. Because of this variability of calving date prediction, it is recommended that ration adjustments such as feed additive or lead feeding, and other management factors such as vaccination should be initiated at least 21 days in advance of expected calving date. Care must be taken, however, to ensure that close-up dry cow rations do not predispose cows to metabolic diseases such as milk fever, udder edema and twisted stomachs.
 

Fastrack® dietary supplementation had no effect (P>0.05) on any of the parameters measured under the conditions of this study (Table 4). In contrast Jaquette et al.(1998) and Ware et al. (1988) reported significantly improved milk production. There was a significantly lower (P<0.05) BCS on cows fed Fastrack® for the study period but this was because on allocation to treatment cows allocated to control scored significantly higher ( P<0.05). There was no significant difference (P>0.05) in change in BCS between treatments over the duration of the study.
 

A covariance analysis of change of BCS based on initial BCS also showed no significant treatment effect (P>0.05). Initial body weight (Table 4) was also slightly higher for control cows but not significant (P>0.05). The combination of higher body weight and condition score of control cows should have increased milk production, but such was not the case. Milk yield was identical (Table 4). Contrary to expectation, the combination of yeast and probiotic did not improve dry matter intake (Table 4). Wohlt et al. (1998) reported significantly improved dry matter intake in their study. Also theorized was that treatment cows might have consumed a larger % of body weight as dry matter because of a slightly lower initial body weight and body condition. Again, no significant difference (P>0.05) was detected (Table 4 and Figure 4).
 
 

Figures 1- 4 show the effects of stage of lactation around calving. Dry matter intake in kg/day and as a % of BW, was lower than anticipated. Wohlt et al. (1998) reported DMI in excess of 24.0 kg/cow/day by the 8th week of lactation whereas in this study intake it was approximately19.0 kg/cow/day at eight weeks. Average intake 2 weeks prior to calving was also low at approximately 10.5 kg/cow/day and 1.45 % of BW. This appears to be similar to data reported by Wohlt et al. (1998), and Robinson (1997). Lead feeding with up to 7.5 -10.0 kg DM of milking cow TMR could leave little opportunity for dry hay consumption and subsequently lead to displaced abomasums.

Although not reported in this paper, there were no differences in metabolic disease occurrences. Effects of Fastrack® on reproductive performance will be reported in another paper.
 

5. Conclusions

In conclusion, Fastrack® fed at 30 g/cow/day from 3 weeks prepartum until 15 weeks postpartum had no effect on cow performance or health. Additional research should be undertaken to evaluate the potential effects of yeast, probiotic combinations during periods of heat stress.
 

7. REFERENCES

Bertics, S.J., Grummer, R.R., Cadorniga - Valino, C. and Stoddard, E.E., 1992. Effect of prepartum dry matter intake on
    liver triglyceride concentration in early lactation. J.Dairy Sci.75: 1914-1922.

Clark, J.H. and Davis, C.L. 1980. Some aspects of feeding high producing cows. J.Dairy Sci. 63: 873-882.

Fuller, R. 1989. Probiotics in man and animal. J . App. Bact. 66:365.

Gill, J.L. 1986. Repeated measurement: senstive tests for experiments with few animals. J.Anim. Sci. 63: 943-954.

Jaquette, R.D., Dennis, R.J., Coalson, J.A., Ware, D.R., Manfredi, E.T., and Read, P.L. 1988. Effect of feeding viable
     Lactobacillus acidophilus (BT 1386) on performance of lactating dairy cows. J. Dairy Sci. 71(Suppl. 1):219.

McGilliard, M.L. and Stallings, C.C. 1998. Increase in milk yield of commercial dairy herds fed a microbial and enzyme
    supplement. J. Dairy Sci. 81:1553-1357.

Robinson, P.H. 1997. Effect of yeast culture on adaptation of cows to diets postpartum.J. Dairy Sci. 80: 1119-1125.

SAS® User's Guide: Statistics, Version 5, Edition -1985    SAS Inst., Inc., Cary, N.C.

Soder, K.J. and Holden, L.A. 1999. Dry matter intake and milk yield and composition of cows fed yeast prepartum and
    postpartum. J. Dairy Sci. 82: 605-610.

Van Saun, R.J., Idleman, S.C. and Sniffen C.J. 1993. Effect of undegradable protein amount fed prepartum on postpartum
    production in first lactation holstein cows.J. Dairy Sci. 76: 236-244.

Ware, D.R., Read, P.L. and Manfredi, E.T. 1988. Lactation performance of two large dairy herds fed Lactobacillus
    acidophilus. J. Dairy Sci. 71 (Suppl. 1): 219.

Wohlt, J.E., Corcione, T.T. and Zajac, P.K. 1998. Effect of yeast on feed intake and performance of cows fed diets based
    on corn silage during early lactation. J. Dairy Sci. 81: 1345-1352.
 
  ______________________________________________________________________________

TABLE 1

INGREDIENT AND CHEMICAL COMPOSITION OF

TOTAL MIXED RATION USED IN THE EXPERIMENT

 



Item                                                                         Percentage of diet *

______________________________________________________________________________

Ingredient

Corn Silage                                                                         29.7

Mixed Haylage                                                                    25.0

High Moisture Corn                                                             27.5

Grain Mix Supplement                                                         17.8

                                                                                           100.0%
 

Chemical Composition

Dry Matter, %                                                                     51.7

Crude Protein, %                                                                16.8

NEL, Mcal/kg                                                                      1.56

ADF, %                                                                              20.3

NDF, %                                                                              31.3

Ca, %                                                                                  0.96

P, %                                                                                   0.51

Mg, %                                                                                 0.44

K, %                                                                                   1.49

______________________________________________________________________________
* Dry Matter Basis
 

______________________________________________________________________________

TABLE 2

COMPOSITION OF GRAIN MIX SUPPLEMENT



 

                                                                                                                % As-FED
 

Roasted soybean                                                                               22.0

Soybean meal (49%)                                                                                 20.0

Corn distillers                                                                       12.5

Corn gluten meal                                                                                   12.0

Canola meal                                                                                   20.0

Cobalt iodized salt                                                                                       3.0

Mineral mix*                                                                                     7.6

Sodium bicarbonate                                                                            2.9

                                                                                                                    100.0

______________________________________________________________________________

*Mineral mix designed to supply 36% calcium, 14% phosphorous, 17% magnesium, 0.6% potassium, 2700 ppm manganese, 1500 ppm copper, 1200 ppm zinc, 500 KIU/kg Vit. A., 220 KIU/kg Vit. D., 2400 IU/kg Vit E and .18 ppm selenium.

______________________________________________________________________________

 

TABLE 3

COMPOSITION OF TOPDRESS SUPPLEMENT



                                                                                                            % As-FED

Soybean meal (49%)                                                                                 24.0

Roasted soybeans                                                                             10.0

Energy booster                                                                                 7.5

Blood meal                                                                                     7.3

Fish meal                                                                                     7.3 Barley                                                                                 25.0

Molasses, wet                                                                                       8.0

Sodium bicarbonate                                                                            3.0

Niacin premix                                                                                  0.4

Limestone                                                                             3.0

Mineral mix*                                                                                     4.5

                                                                                                            100.0

______________________________________________________________________________

*Mineral mix designed to supply 22.7% calcium, 8.5% phosphorous, 3.0% magnesium, 1830 ppm manganese, 510 ppm copper, 1630 ppm zinc, 45 KIU/kg Vit. A., 5 KIU/kg Vit. D., 68 IU/kg Vit. E and 0.56.ppm. selenium.

 

Table 4

 

EFFECTS OF PROBIOTIC YEAST SUPPLEMENT ON PERFORMANCE OF TRANSITION DAIRY COWS
 

 

Control

Treatment

+ SEM*

Body Weight (BW), kg

637

613

19.7

Body Weight Change, kg

-77

-77

34.5

Dry Matter Intake, kg

18.0

17.6

1.05

DMI, % of BW

2.86

2.92

0.19

BCS

2.60b

2.07a

0.12

Milk Yield, kg

40.9

40.9

2.4

Somatic Cell Count, 000's ml

98.0

206.3

54.1 

Milk Urea Nitrogen, mg%

14.9

15.1

0.44

Milk Fat, %

3.58

3.63

0.11

Milk Protein, %

2.84

2.92

0.05

* SEM: Standard error of least square means

a,b. Least square means in the same row with different postscripts are significantly different (P<0.05)
 
 

For more information, please contact:  dmcknigh@kemptvillec.uoguelph.ca
 
 
 
 
 
 7. REFERENCES

Bertics, S.J., Grummer, R.R., Cadorniga - Valino, C. and Stoddard, E.E., 1992. Effect of prepartum dry matter intake on
    liver triglyceride concentration in early lactation. J.Dairy Sci.75: 1914-1922.

Clark, J.H. and Davis, C.L. 1980. Some aspects of feeding high producing cows. J.Dairy Sci. 63: 873-882.

Fuller, R. 1989. Probiotics in man and animal. J . App. Bact. 66:365.

Gill, J.L. 1986. Repeated measurement: senstive tests for experiments with few animals. J.Anim. Sci. 63: 943-954.

Jaquette, R.D., Dennis, R.J., Coalson, J.A., Ware, D.R., Manfredi, E.T., and Read, P.L. 1988. Effect of feeding viable
     Lactobacillus acidophilus (BT 1386) on performance of lactating dairy cows. J. Dairy Sci. 71(Suppl. 1):219.

McGilliard, M.L. and Stallings, C.C. 1998. Increase in milk yield of commercial dairy herds fed a microbial and enzyme
    supplement. J. Dairy Sci. 81:1553-1357.

Robinson, P.H. 1997. Effect of yeast culture on adaptation of cows to diets postpartum.J. Dairy Sci. 80: 1119-1125.

SAS® User's Guide: Statistics, Version 5, Edition -1985    SAS Inst., Inc., Cary, N.C.

Soder, K.J. and Holden, L.A. 1999. Dry matter intake and milk yield and composition of cows fed yeast prepartum and
    postpartum. J. Dairy Sci. 82: 605-610.

Van Saun, R.J., Idleman, S.C. and Sniffen C.J. 1993. Effect of undegradable protein amount fed prepartum on postpartum
    production in first lactation holstein cows.J. Dairy Sci. 76: 236-244.

Ware, D.R., Read, P.L. and Manfredi, E.T. 1988. Lactation performance of two large dairy herds fed Lactobacillus
    acidophilus. J. Dairy Sci. 71 (Suppl. 1): 219.

Wohlt, J.E., Corcione, T.T. and Zajac, P.K. 1998. Effect of yeast on feed intake and performance of cows fed diets based
    on corn silage during early lactation. J. Dairy Sci. 81: 1345-1352.
  
  ______________________________________________________________________________

TABLE 1

INGREDIENT AND CHEMICAL COMPOSITION OF

TOTAL MIXED RATION USED IN THE EXPERIMENT

 



Item                                                                         Percentage of diet *

______________________________________________________________________________

Ingredient

Corn Silage                                                                         29.7

Mixed Haylage                                                                    25.0

High Moisture Corn                                                             27.5

Grain Mix Supplement                                                         17.8

                                                                                           100.0%
 

Chemical Composition

Dry Matter, %                                                                     51.7

Crude Protein, %                                                                16.8

NEL, Mcal/kg                                                                      1.56

ADF, %                                                                              20.3

NDF, %                                                                              31.3

Ca, %                                                                                  0.96

P, %                                                                                   0.51

Mg, %                                                                                 0.44

K, %                                                                                   1.49

______________________________________________________________________________
* Dry Matter Basis
 

______________________________________________________________________________

TABLE 2

COMPOSITION OF GRAIN MIX SUPPLEMENT



 

                                                                                                                % As-FED
 

Roasted soybean                                                                              22.0

Soybean meal (49%)                                                                                 20.0

Corn distillers                                                                       12.5

Corn gluten meal                                                                                   12.0

Canola meal                                                                                   20.0

Cobalt iodized salt                                                                                      3.0

Mineral mix*                                                                                     7.6

Sodium bicarbonate                                                                            2.9

                                                                                                                    100.0

______________________________________________________________________________

*Mineral mix designed to supply 36% calcium, 14% phosphorous, 17% magnesium, 0.6% potassium, 2700 ppm manganese, 1500 ppm copper, 1200 ppm zinc, 500 KIU/kg Vit. A., 220 KIU/kg Vit. D., 2400 IU/kg Vit E and .18 ppm selenium.

______________________________________________________________________________

 

TABLE 3

COMPOSITION OF TOPDRESS SUPPLEMENT



                                                                                                            % As-FED

Soybean meal (49%)                                                                                 24.0

Roasted soybeans                                                                             10.0

Energy booster                                                                                 7.5

Blood meal                                                                                     7.3

Fish meal                                                                              7.3

Barley                                                                                 25.0

Molasses, wet                                                                                       8.0

Sodium bicarbonate                                                                           3.0

Niacin premix                                                                                  0.4

Limestone 

                                                                                            3.0

Mineral mix*                                                                                     4.5

                                                                                                            100.0

______________________________________________________________________________

*Mineral mix designed to supply 22.7% calcium, 8.5% phosphorous, 3.0% magnesium, 1830 ppm manganese, 510 ppm copper, 1630 ppm zinc, 45 KIU/kg Vit. A., 5 KIU/kg Vit. D., 68 IU/kg Vit. E and 0.56.ppm. selenium.

 

Table 4

 

EFFECTS OF PROBIOTIC YEAST SUPPLEMENT ON PERFORMANCE OF TRANSITION DAIRY COWS
 

 

Control

Treatment

+ SEM*

Body Weight (BW), kg

637

613

19.7

Body Weight Change, kg

-77

-77

34.5

Dry Matter Intake, kg

18.0

17.6

1.05

DMI, % of BW

2.86

2.92

0.19

BCS

2.60b

2.07a

0.12

Milk Yield, kg

40.9

40.9

2.4

Somatic Cell Count, 000's ml

98.0

206.3

54.1 

Milk Urea Nitrogen, mg%

14.9

15.1

0.44

Milk Fat, %

3.58

3.63

0.11

Milk Protein, %

2.84

2.92

0.05

* SEM: Standard error of least square means

a,b. Least square means in the same row with different postscripts are significantly different (P<0.05)