Preliminary trials of a specific gravity technique in the determination of early embryo growth potential†

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Published on: July 26, 2019

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S.D. Prien,1,2,* C.E. Wessels,2 and L.L. Penrose1

. 2015 Sep; 30(9): 2076–2083.
Published online 2015 Jul 22. doi: 10.1093/humrep/dev178
PMCID: PMC4542720
PMID: 26202920

Abstract

STUDY QUESTION

Can a modified specific gravity technique be used to distinguish viable from nonviable embryos?

SUMMARY ANSWER

Preliminary data suggests a modified specific gravity technique can be used to determine embryo viability and potential for future development.

WHAT IS KNOWN ALREADY

Single embryo transfer (SET) is fast becoming the standard of practice. However, there is currently no reliable method to ensure development of the embryo transferred.

STUDY DESIGN, SIZE, DURATION

A preliminary, animal-based in vitro study of specific gravity as a predictor of embryo development using a mouse model.

PARTICIPANTS/MATERIALS, SETTING, METHODS

After a brief study to demonstrate embryo recovery, experiments were conducted to assess the ability of the specific gravity system (SGS) to distinguish between viable and nonviable embryos. In the first study, 1-cell mouse embryos were exposed to the SGS with or without previous exposure to an extreme heat source (60°C); measurements were repeated daily for 5 days. In the second experiment, larger pools of 1-cell embryos were either placed directly in culture or passed through the SGS and then placed in culture and monitored for 4 days.

MAIN RESULTS AND THE ROLE OF CHANCE

In the first experiment, viable embryos demonstrated a predictable pattern of descent time over the first 48 h of development (similar to previous experience with the SGS), while embryos that were heat killed demonstrated significantly altered drop patterns (P < 0.001); first descending faster. In the second experiment, average descent times were different for embryos that stalled early versus those that developed to blastocyst (P < 0.001). Interestingly, more embryos dropped through the SGS developed to blastocyst than the culture control (P < 0.01).

LIMITATIONS, REASONS FOR CAUTION

As this is a preliminary report of the SGS technology determining viability, a larger embryo population will be needed. Further, the current in vitro study will need to be followed by fecundity studies prior to application to a human population.

WIDER IMPLICATIONS OF THE FINDINGS

If proven, the SGS would provide a noninvasive means of assessing embryos prior to transfer after assisted reproductive technologies procedures, thereby improving fecundity and allowing more reliable SET.

STUDY FUNDING/COMPETING INTEREST(S)

The authors gratefully acknowledge the funding support of the U.S. Jersey Association, the Laura W. Bush Institute for Women’s Health and a Howard Hughes Medical Institute grant through the Undergraduate Science Education Program to Texas Tech University. None of the authors have any conflict of interest regarding this work.

TRIAL REGISTRATION NUMBER

none.

Keywords: embryo development, embryo selection, embryo viability, specific gravity, buoyance, noninvasive, zygote, blastocyst

Legality of Compounded Estradiol for Embryo Transfer

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Published on: July 25, 2019

The AABP office has received questions from members regarding the legality of using compounded estradiol products in cattle for embryo transfer protocols. AABP has also been in discussion with the FDA about the use of compounded estradiol products in food animals. Compounding from approved drugs in animals is only permitted under the narrowly defined conditions outlined in AMDUCA (Section 21 CFR 530.13). To be permitted, extralabel use from compounding of approved animal drugs or approved human drugs must be in compliance with all relevant provisions of 21 CFR 530 (AMDUCA), including the provisions limiting extralabel use to treatment modalities when the health of an animal is threatened or suffering or death may result from failure to treat. The extralabel use regulation also does not provide for compounding from active pharmaceutical ingredients (APIs or bulk drugs—i.e., the raw chemical) for use in animals. Therefore, it is illegal for veterinarians to use or prescribe compounded estradiol for cattle, or any form of estrogenic compounds for production purposes, including embryo transfer and synchronization protocols. AABP encourages cattle veterinarians to refrain from administering or prescribing compounded estradiol for the following reasons:

  • AMDUCA only allows for extralabel drug use when the health of an animal is threatened. There is no production allowance, particularly for compounding; therefore one cannot use human-approved drugs (e.g., ECP, Pfizer) or a different form of an animal-approved drug (e.g., growth-promoting implants) for production purposes.
  • Compounding from a bulk product is specifically prohibited in AMDUCA regulations.
  • The safety, potency, efficacy, stability, sterility, and disposition of compounded products is unknown. Compounded products do not undergo FDA inspection, potency testing, or efficacy testing. Veterinary compounding pharmacies that also compound for humans are under federal regulation and are FDA inspected; however, this only applies to the human side of the compounding operation. Veterinary compounding pharmacies do not have this level of oversight. There is no guarantee of the safety or efficacy of compounded products, and liability for the use of such products falls on the veterinarian in the event of an adverse reaction or violative residue.
  • Because the safety, efficacy, potency, and disposition of the compounded product is not known, it is impossible to assign a withdrawal interval for compounded products.
  • The use of compounded products in food animals places a veterinarian at risk of professional liability.

The need for estradiol for successful embryo transfer protocols has not been unequivocally established. For example, data from nearly 7,000 collections did not demonstrate a difference when using GnRH in place of estradiol in the protocol.1 Additional references are available on the Reproduction Committee page of the website at https://aabp.org/members/Reproduction.asp. Veterinarians who engage in federally prohibited activities put themselves at risk and also risk the profession’s reputation for appropriate and judicious oversight of pharmaceutical products in our cattle patients. This is especially of concern when using an unapproved and illegally manufactured hormone product.

AABP Newsletter 5 May 2019.

Please contact Dr. Fred Gingrich at fred@aabp.org with any questions.

Submitted by the AABP Reproduction Committee and the AABP Committee on Pharmaceuticals and Biologics.

1Hinshaw, R.H. Comparison of GnRH and estradiol 17β for follicle turnover in bovine superovulation protocols. Proceedings of the American Embryo Transfer Association 2013, p. 15.

AETA Small Ruminant Ovum-Pick-Up Brief

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Published on: April 17, 2019

Submitted by Dr. Kevin Lindell

Recently, Dr. Rachael Gately, Tufts Veterinary Field Service, had the opportunity (through a collaborative research project) to explore the realm of small ruminant ovum pick-up (OPU)/in vitro fertilization (IVF).

We thought it would be interesting to the membership to relay our initial experiences and challenges.

To date, donors have been collected after superstimulation, using protocols somewhat similar to those we use in cattle. Without prior manipulation of the estrus cycle, a SR CIDR is placed on day −7. On day 0 donors receive PG and 1.5 cc of follicle-stimulating hormone (FSH) AM/PM; day 1 donors receive 1.0 cc of FSH AM/PM and then 1.0 cc of FSH on the morning of day 2. Oocyte collection is performed on day 4, resulting in a coasting period (time between the last FSH injection and time of collection) of approximately 48 hours. Variables such as FSH dosage, number of total injections, and coasting period seemed to make a significant impact on follicular recruitment, size, and competency, although the data set is very small to this point.

Oocyte collection is performed under general anesthesia using a short-needle system. A threaded small ruminant 18-ga OPU needle, attached directly to aspiration tubing (without using a metal rod) has been a relatively simple and successful method for oocyte collection, in conjunction with our regular aspiration/vacuum pump. Although we would ultimately like to offer small ruminant OPU as a purely laparoscopic procedure, it has been difficult to stabilize the ovaries well enough, so we have instead opted to externalize the ovaries briefly for aspiration.

As this procedure seems to be growing in interest amongst our clientele, we are looking forward to additional small ruminant oocyte collection trials and embryo development results.

The abstract below from a recent review article was also a useful tool as we initially organized equipment, consumables, and donor protocols.

Theriogenology (86) 2016

Recent advances in in vitro embryo production in small ruminants

By Maria-Teresa Paramio*, Dolors Izquierdo

ABSTRACT

To increase productivity in the small ruminant industry, the genetic material of these species should be improved. In vitro embryo production could be an important technology to reach this goal by combining selected male and female gametes. In the world, marketing of in vitro-produced embryos is an economical activity which is progressing rapidly in cattle but is practically nonexistent in small ruminants. Since the birth of the first lamb and kid using IVF in the 80s, several studies have been carried out; however, results still are inconsistent and unpredictable. Moreover, significantly fewer research groups are working on embryo production in small ruminants than in cattle and pigs. Although conventional methodologies of oocyte IVM, IVF, and IVC in small ruminants give rise to blastocysts, significant variation exists between experiments. One important reason for these differences is the heterogeneity of the pool of oocytes recovered from ovaries from slaughtered females. Oocyte quality, also referred to as competence, is the key factor in the success of in vitro embryo production programs. Different criteria are used to select the best oocytes for fertilization, such as follicle size, oocyte diameter and morphological appearance, and Brilliant Cresyl Blue staining. New research lines aimed at improving oocyte competence are: (1) arresting nuclear maturation in vitro allowing optimal capacitation of cytoplasm, (2) growing oocytes inside the follicle, and (3) identification of biomarkers of oocyte competence in granulosa and cumulus cells and metabolites in the follicular fluid.

Practice Tip – Battery Backup

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Published on: April 17, 2019

Submitted by Dr. Pat Comyn

This jump starter has come in really handy for those times when I am breeding or placing embryos in a place with no power. With all the moisture we have had in my area in the last year (92 inches), it is sometimes really difficult to get a truck near the working area. Also, trucks near the working area = dented trucks. So this gadget is great for keeping the thaw bath warm. It also charges cell phones.  

 

New Module on Frozen Semen Evaluation Available

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Published on: December 27, 2018

The AETA Education Committee has partnered with Brad Stroud, DVM, and is pleased to announce that a new educational module is available to AETA members.

To access the new Frozen Semen Spot Test, please log in to the AETA site and follow this link: https://www.aeta.org/edu-teaching-module-frozen-semen.asp. You can view the module in your browser, on your phone, or download it (it is a large file).

Thank you to Brad Stroud, DVM.

 

Practice Tip

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Published on: July 6, 2018

by Pay Comyn
AETA Education Committee Chair

I saw a good reminder of dangers of using frozen cold pacs to chill down fresh transported embryos. A client accidentally froze several syringes of rabies vaccine meant for their cattle by placing the syringes directly on frozen ice packs. So embryo straws with holding solution placed directly on frozen ice packs would be risky. When using frozen ice packs to keep fresh embryos cool, one should place cloth or paper over the ice packs to protect the embryos.

Effects of Ovum Pick‐up Frequency and FSH Stimulation: A Retrospective Study on Seven Years of Beef Cattle In Vitro Embryo Production

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Published on: July 6, 2018

Authors: R De Roover, JMN Feuganf, PEJ Bois, G Genicot, Ch Hanzen

Publication: Reproduction in Domestic Animals

Publisher: John Wiley and Sons

Date: Mar 6, 2008

Link: https://doi.org/10.1111/j.1439-0531.2007.00873.x

My IVF incubator is late….now what?

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Published on: April 10, 2018

By Jon Schmidt (Trans Ova Genetics)

An unfortunate reality with IVF is the occasional need to utilize commercial shipping companies in the transportation of oocytes to and embryos from the IVF lab.  Many of us who work with IVF shipments have experienced a delayed, lost, or cold incubator.  These are unfortunate events that can be catastrophic to results and end in frustrated lab staff, transfer teams, and clients.  Below are a few suggestions on how to handle incubators that are compromised in transit while embryos are going back to the practitioner or client’s farm.

d0 = OPU day
d1 = fertilization day
d7 = normal transfer day

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AETA Practice Tip: Placing CIDR

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Published on: April 10, 2018

By Tyler Dohlman (Iowa State University)

For most of us practitioners, CIDR have become a staple in our reproductive programs and will continue to be as long as they are effective. Whether they are used on donors or recipients or in artificial insemination protocols, they allow us to manipulate the estrous cycle for various needs. However, everyone has had a time when the number of CIDR put in does not match the number found when it comes time to retrieve them. This scenario can happen in a few instances: (1) we did not actually place a CIDR in the first place or (2) they fell out or were pulled out prior. CIDR, in general, should not come out sporadically, and if they do, it is usually because of poor or haphazard placement. Therefore, the latter scenario in which they are getting pulled out is more likely. In our experience, heifers are the problem child group. Heifers, curious in nature, and especially Holsteins, are all too accustomed to making our lives much more difficult by helping us pull CIDR out before our protocol says.

To mitigate this issue, I was taught at some point in my career to clip the blue attached string short. However, I usually forget to grab scissors or a knife to do such modifications to the CIDR at placement. Conveniently enough, I was taught a different modification to hide that all-too-enticing blue string. The modification is simple and easy, and rarely, if ever, do we lose CIDR in those curious Holstein heifers anymore.

If you have ever looked at a CIDR, there is a hole the same size as the blue string on the base of the CIDR. All you have to do is flip the blue string in the hole before placing the CIDR in the applicator. This hides the blue string and conveniently creates a looped handle for removal. In our hands, this modification has worked on our farms with heifers. Clients are less adaptive to this new method because they cannot see the blue string to confirm the CIDR is still in place, but the persuasive nature in me explains that if a CIDR were to fall out prior to protocol, it could prevent an ET or AI pregnancy. Caution: we commonly do this in cows also, and sometimes the blue string handle is in just a little too far and is out of reach. Then trans-rectal palpation guidance is needed to push the CIDR closure to the vulvar opening for retrieval.

On that note, I hope this helps someone and that you never lose another CIDR.

8 Questions You May Have About Cryopreserving Bovine In Vivo–Derived Embryos

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Published on: December 27, 2017

John F. Hasler
Jfhasler05@msn.com
John.hasler@vetoqinol.com
Cell: 970-222-5302

Dr. Pat Comyn, the new chair of the AETA Education Committee, asked me to write a short piece clarifying some issues concerning the cryopreservation of bovine embryos for inclusion in the December issue of A Closer Look. The AETA has come a long way since our humble beginnings in 1983, and our 2017 membership now totals 556, including a large increase in the number of new members. The following facts and suggestions will be of most interest to our new and less experienced members. Not only are there many variables involved in successfully freezing and thawing bovine embryos, there also are many variations on most of the steps that do not notably detract from success rates. Having worked with many ET practitioners in 17 different states and a number of foreign countries, I have a pretty good idea of what works well and what does not. The following points are either based on published data that I deem to be replicable or based on my own experience and observations. Please feel free to contact me should you want advice or clarification.

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VitaFerm Article: Preparing Cows for Embryo Transfer

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Published on: December 27, 2017

Prepare Cows for Embryo Transfer

Embryo transfer (E.T.) is an important tool to propagate outstanding genetic influence within the herd, with the potential to produce multiple offspring of the same mating in the same year. Because of the time, labor and expense involved in creating these genetics, we reached out to Trans Ova Genetics, a leader in reproductive technologies, to provide useful information to prepare your cows for a successful E.T. program.

According to Trans Ova, successful E.T. programs require intensive management and attention to detail. The results you achieve are highly variable and the level of success is based on your ability to manage all aspects of the operation.

Get Your Recips Ready

Preparing recipient cows for their role of carrying and growing the embryo is not a lot different than preparing cows to be bred naturally. You want to keep them in a low-stress environment, be consistent in daily management practices, give all vaccinations prior to estrus and make sure their nutrition program is supplemented with high levels of trace minerals like copper, zinc and manganese that impact reproductive success.

“Nutrition is without a doubt one of the most important areas of donor and recipient management,” said Jon Schmidt, DVM and Chief Operations Officer at Trans Ova. “First of all, I believe the nutritional management of your cattle needs to be a year-long process. Attention should be placed on meeting their demands for the entire season including gestation and lactation.”

The most critical and demanding time however, includes the month before calving through the first three to four months after calving. This is the most stressful and nutritionally demanding time to allow that cow to produce a healthy calf via colostrum production, begin lactation to raise that calf and become pregnant.

Reproduction is not an essential process in survivability of that cow, and consequently suffers first if nutritional needs are not met. Maintenance and milk production will partition available energy supplies with reproduction suffering at their expense. Therefore, it is critical to meet their requirements. Ensure cows are fed a high-quality mineral especially one that optimizes zinc, selenium and copper as they are critical for successful embryo transfer outcomes. Avoid rations that are high in distiller’s byproducts or sulfur-containing forages. Avoid diets high in Urea.

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The Role of Trace Minerals in Beef Cattle Fertility

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Published on: December 27, 2017

Ratzburg, Cole, The Role of Trace Minerals in Beef Cattle Fertility, M.S., Department of Animal Science, August 2017.

The most important economic factor influenced by trace mineral deficiencies is impaired reproductive performance in both the male and female. Copper (Cu), zinc (Zn), and manganese (Mn) have been shown to improve fertility in male and female beef cattle due to their roles in vitamin synthesis, hormone production, enzyme activity, collagen formation, tissue synthesis, oxygen transport, energy production, and other physiological processes related to growth, reproduction, and health. A new form of trace minerals called hydroxy minerals, which is a hydrolyzed inorganic metal complex, has been shown to have a greater bioavailability than sulfate minerals and similar bioavailability compared with the organic trace minerals. The idea that hydroxy trace minerals could be more available to beef cattle led us to hypothesize that use of hydroxy forms of Cu, Zn, and Mn could improve fertility in male and female beef cattle that are transitioning into the pubertal phase. The objectives of the two studies were to determine whether the use of hydroxy trace minerals could improve fertility parameters related to both the male and female beef cow. Peripubertal bulls were supplemented with hydroxy forms of Cu or Zn or Cu and Zn or no Cu and Zn in an 83-day mineral trial to determine whether there were differences in liver, blood plasma, and semen mineral concentrations and subsequently breeding soundness exam parameters, sperm morphology, and flow cytometer analysis. Results showed the use of Cu and Zn had benefits for fertility; there was improvements in flow cytometer parameters and sperm morphology. Heifers were supplemented with sulfate and hydroxy forms of Cu, Zn, and Mn to determine whether the different forms of trace minerals could affect feed intake parameters such as average daily gain, dry matter intake, residual feed intake, and feed-to-gain ratio and the fertility parameters: percent cycling and percent conception. Results indicated that there were no differences in feed intake parameters between treatments, but there was a positive benefit observed with the hydroxy trace minerals on conception percentage.

 

Bovine in vitro embryo production – What happens after OPU?

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Published on: September 26, 2017

AETA Practice Tip

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Published on: September 26, 2017

By Marianna Jahnke and Dr. Tyler Dohlman, College of Veterinary Medicine, Iowa State University

Who has not gotten frustrated with a straw exploding or issues with loading a direct transfer 0.25-cc straw? Last year we had some problems with the yellow 0.25-cc DT straws that are commercially sterilized and repackaged in groups. We had a few straws exploding, and within those straws we noticed that the cotton plugs and the PVC were separating during the loading process. As we were pulling the last column of ethylene glycol, the ethylene glycol kept going through the cotton plugs into the syringe as seen on the images. As we all have been trained and continue to train others, it is important to make sure fluid penetrates the PVC and cotton plugs in order to create a good seal to the straw. With this issue, our concerns were that the straws were not sealing properly, leaving them at an increased risk of exploding during the thawing process or even potentially allowing liquid nitrogen to enter the straw. This is probably not new information to some of you who have been in the industry for some time and it probably has been discussed over the years, but this was our first major straw issue. In addition, we have noticed other straws from other ET practitioners that seem to be doing the same thing, with a cotton plug being exposed at the end of the straw or gone completely.

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Evidence-based ET: Is the exposure time of bovine embryos to ethylene glycol (EG) prior to freezing and/or after thawing critical to survival?

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Published on: September 26, 2017

Is the exposure time of bovine embryos to ethylene glycol (EG) prior to freezing and/or after thawing critical to survival?

John F. Hasler

Efficient and efficacious cryopreservation of bovine embryos is critical to the commercial ET industry because, as shown by the most recent AETA statistics (2011), 72% of embryos were frozen following collection versus only 28% that were transferred fresh into recipients. Following the published report of Voelkel and Hu in 1992 on cryopreservation with EG, the commercial bovine ET industry rather quickly switched from glycerol to EG as the major cryoprotectant in freezing media. The overall percentage of embryos frozen in EG rose rapidly starting in 1992 and reached 97% in 2008, the last year that the AETA collected data on this specific statistic.

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Small Dry Shippers

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Published on: September 26, 2017

By Karen Rockey

Let’s face it, without our cryogenic tanks, we would not be in business. So let’s take a little time to discuss the tanks we use for export shipping of embryos.

Small dry shippers and Dobles are most widely used for domestic transportation and shipping to neighboring countries, because of their size and non hazardous status. Unfortunately, they have limited hold times. Knowing the hold time is very important. If you have issues in customs, or papers are lost, a hold time of 14 – 17 days doesn’t give you much time to rectify the situation.

One of the items available (and recommended for all wide mouth dry shippers), is the cabosil insert. When fully absorbed with nitrogen and placed inside the tank with the embryos, it will give the tank a little extra time of charge. Insurance may also require the use of these inserts. I am not sure if others are making these now, but our source has been Chart/MVE.

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Understanding Shoulder Pain and How to Return to Living

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Published on: September 26, 2017

By Meredith Griffin PT, DPT

Shoulder pain is a very common issue. Every day in the clinic, I see at least one patient with complaints of shoulder pain and the only other diagnosis I see more frequently is low back pain. Shoulder pain can be brought on by a variety of causes, including over use, trauma, and aging. Veterinarians, especially ET practitioners, are highly susceptible to the former of the three. Despite the cause, the progression of therapy is generally the same.

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Sleep Deprivation

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Published on: September 26, 2017

By Mid-Maryland Vets

I had a weird experience at an early morning herd check.  Donald was bouncing off the walls, talking a mile a minute, yelling, and sprinting like a mad man.  He bragged about the all-nighter he spent out in the fields before coming home to milk and prep for herd check.  He had popped a few caffeine pills chased with black coffee an hour before to amp him up for the morning which explained his nuclear energy level.  I think I could even see his heart trying to explode out of his chest. A visit to another farmer during late spring revealed a completely run-down, exhausted, incoherent mess of a man barely able to function.  But like the aforementioned spastic man, this fellow also bragged about his lack of sleep.  Most farmers and a few veterinarians love to brag about the all-nighters and present their lack of sleep like a badge of honor.  They believe that pushing through exhaustion by extending the work day and depriving themselves of sleep makes them more productive.  In reality, sleep deprivation makes us less productive.

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Summer’s Heat Stroke

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Published on: June 30, 2017

By Mid Maryland Dairy Veterinarians

Summer is finally here!  With summer upon us, the Mid-Atlantic region can look forward to the 3 H’s:  Hazy, Hot, and Humid.  We should not be surprised to witness one of those epic heat waves with scorching hot and dangerously high heat indexes close to 110F. Heat stroke is a dangerous condition that cattle can experience during a heat wave.  Do we need an epic heat wave to create heat stroke?  Absolutely not!  Exertion from calving, disease, and poor facility ventilation/heat abatement can induce heat stroke.  You need to be able to recognize the animal experiencing heat stroke, treat it quickly, and take steps to minimize the risk of heat stress.

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Exporting Efficiency

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Published on: June 30, 2017

EXPORTING EFFICIENCY

Are your records accurate?

QUARANTINE!

We’ve all heard it, and most have experienced it in one way or another.

As traceability becomes more of an issue, registration numbers are very important. Complete registration numbers; leaving zeros out of an 840 registration number will land you in quarantine. Forgetting the CAN in front of a Canadian registration number or USA in front of the USA registration numbers can end up in quarantine as well. Including official ear tag numbers on testing is also important and required by the USDA.

Exporters ask for a D form copy to make sure the numbers and names are correct: Exactly correct. It is a simple task to verify this information on the individual breed website. This is the first thing done in the export preparation process. Holstein (http://www.holsteinusa.com/hol/animalSearch.action), Jersey, Angus, Hereford, and Wagyu, to name a few, all have easily accessible websites. Leaving out an –ET can be as much of a problem as unnecessarily adding it. Misspelling is another common issue, especially with shortened registered names. For the Holstein 840 numbers, make sure it matches the official pedigree (84000…), even though it is shown at least three different ways on their website.

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