I suspect that as you read this newsletter you may have some of the same feelings that I have. I am finding that the summer is flying by and I am not sure where June and July went. Many of us spend a great deal of time looking out our windshield as we move down the road every day. My thoughts have been that these are interesting times that we live in.
Many of you have reached out to the AETA board, and I appreciate that. We have taken your thoughts and concerns and tried to create lemonade from lemons this year. Soon, information regarding registration for this year’s joint virtual conference will be on the AETA website with details about the schedule and content. Our intention is to deliver meaningful continuing education opportunities as well as satisfy the certification requirement with flexibility at a reasonable cost.
We will have sessions available for downloading at your convenience as well as three sessions that will be offered live, including chat interaction with the speaker. These three live sessions will be recorded for viewing at your convenience as well. There will also be a virtual business meeting that will include the election of board members. I encourage you all to try to make the business meeting a priority for attendance.
The registration fees for the virtual conference reflect the board’s efforts to control cost and pass that value on to the membership and sponsors. These are uncertain times and we do not want the cost of the meeting to be a barrier if possible. The AETA board is also mindful of the fact that this could be an opportunity to reach new members on a different platform. With that in mind, we will offer the option to apply the registration fee for the 2020 virtual conference to an equivalent discount for 2021 AETA membership if you desire. Please share the word with anyone who might be interested.
The last aspect of the revised convention proceedings I want to discuss is a change in certification requirements. The board has changed the requirements for conference attendance to 3 out of 5 years, which was previously 2 out of 3 years. This recommendation from the certification committee is meant to reflect the logistics related to this year’s virtual convention as well as the convention in Canada next year. We are also waiving the newly established “in-person” recertification that was to be introduced this year and was meant to replace the on-site random recertification inspections.
This year’s virtual proceedings are an opportunity to try something new and see what happens. I look forward to your participation.
The 2020 AETA-CETA/ACTE Joint Annual Meeting will be held virtually beginning on October 6th. You must register before October 6th to get access to the meeting content.
All of the scientific, sponsor, and CE information can be found on the AETA Annual Convention page as it becomes available. Check back often!
The convention will feature two types of sessions: keynote addresses and prerecorded sessions.
Keynote addresses will be live sessions via Zoom, where participants will see the content and interact with the speakers in real time. Once the keynote addresses have concluded, a link to the content will be posted.
Prerecorded sessions will be made available via a link on the AETA convention page at 12:01 am (CDT) on Tuesday, October 6. During the times on the schedule, speakers will be available to chat via Zoom about their presentations and answer any questions you may have.
All sessions will have a quiz that participants must participate in and pass with a score of at least 70% to receive continuing education (CE) credits.
Please note that speakers will only be available for discussion during their scheduled time. Participants who register before October 6th can view sessions and take the accompanying RACE quizzes until December 31st.
You must register before October 6th to get access to the meeting content.
The AETA Board of Directors voted to remove the requirement for random inspections of certified members from the current guidelines, starting immediately:
IV. CERTIFICATION PROGRAM ADMINISTRATION
D. Inspection and Noncompliance
2. RANDOM: Inspections of any certified practitioner’s ETB may be done at any time under the direction of the Board of Directors and the CAO.
and replace it with a NEW requirement (that would not start until the fall meeting in 2021):
D. Inspection, Compliance, and Noncompliance
2.CERTIFICATION SESSION: 20% of certified members will be required to attend a mandatory group certification session at the annual meeting. Thus, every certified member would attend one session during his/her cycle.
2020 CERTIFICATION EXAM AND EXAM RETAKE UPDATE
People seeking to sit for the 2020 Certification Exam and Practicum will still be able to voluntarily do so this year at the Iowa State University (ISU) College of Veterinary Medicine in Ames, Iowa, on Saturday, September 26,in person. There may be a limit to the number of examinees due to ISU restrictions on groups, plus examinees will be asked to sign waivers of liability to hold harmless the host and the AETA.
Any recent certification candidate that needs to retake a portion(s) of the certification exam needs to contact AETA headquarters (aeta@assochq.org) or Glenn Engelland (glennengelland@gmail.com), Certification Committee chair, to schedule a live Zoom conference retake.
Elections will take place during the Annual Business Meeting scheduled for October 7 from 1:30pm-2:30pm central time. Please take a moment to read the candidates bios below.
So now you can buy that fancy tall stainless steel thermal mug and call it a business expense! These vacuum mugs hold liquid nitrogen (and do so, like a dewar) without the outside getting cold. Great for caning embryos, recaning semen, treating cancer eye, etc.
When exporting beef embryos, a lot of the CSS collected semen is done by small bull studs. These outfits often are unfamiliar with embryo export requirements so be sure to check the CSS semen certificate from these centers to ensure that an accredited veterinarian has signed the CSS document. A non DVM, etc, signature is not valid.
Dalena Hobbs, Colton Holcomb, and John Gibbons, College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN, 37752
Introduction:
Embryo transfer is an assisted reproductive technique that enables progressive cattle producers to reach their financial, reproductive, and genetic goals and the process began to gain considerable traction in the late 1970s to early 1980s as non-surgical methods to collect embryos were developed (Troxel, 2013). Currently, conventional embryo collection techniques require ovarian hyper-stimulation of donor cows with exogenous FSH, artificial insemination, and trans-cervical uterine lavage to recover embryos about 7 days post insemination. Another approach that has gained popularity is in vitro fertilization (Mapletoft, 2013). In vitro embryo production involves recovering the ova directly from the ovaries using an ultrasound guided trans-vaginal follicular aspiration technique. The recovered oocytes are matured, fertilized, and cultured in vitro and this approach has become substantially popular recently despite the increased cost associated with specialized equipment, training, staff, etc. According to AETA reports (AETA, 2017; 2019) there has been a 120% increase in the number of in vitro produced embryos transferred over the most recent two years of available AETA data (AETA, 2017; 2019). In vitro embryo production seems to be gaining popularity over conventional embryo transfer techniques because of the potential to produce more calves per year (Stadheim, 2015), may require fewer hormone injections, and does not require synchronization. In conventional in vivo embryo collections, almost one half of recovered ova are non-viable, and that percentage has not changed substantially in many years (AETA, 2010; 2019). Many of these non-viable ova are considered degenerate embryos that have not developed to the appropriate stage relative to the other embryos in the cohort. Salvaging these degenerate embryos that would otherwise be discarded may translate to additional embryo transfers or calves per embryo collection. This experiment evaluated short term incubation environments and the potential damage to the embryo and zona pellucida associated with the freeze / thaw process. As many practitioners have become involved in in vitro embryo production, the equipment, supplies, and staff are likely in place to consider an in vitro culture approach of degenerate or poor quality embryos (fresh or post-thaw) to enable the development of these embryos.
Methods and Materials:
Frozen / thawed bovine (Day 7) in vivo derived embryos processed for direct transfer (Ethylene Glycol) were thawed for 30 seconds in a 30°C water bath. Thawed embryos (total of 30 / group over three replicates) were placed in commercially available holding media temporarily to be graded and staged (according to the International Embryo Technology Society rubric) and then placed into either holding media, phosphate buffered saline (PBS) supplemented with 15% fetal bovine serum (FBS; v/v) and antibiotic / antimycotic (gentamicin; 2 mL/ml; v/v), or a commercially available in vitro culture media for approximately 18 hours at 38.5°C. Embryos in the holding media and PBS (+15% FBS) were loaded (individually) into ¼ cc plastic straws which were sealed and submerged in a water bath (38.5°C). Embryos in the in vitro culture media group were rinsed and placed (individually) in equilibrated 25 mL culture drops on tissue coated plastic 60 mm dishes overlaid with lightweight mineral oil and were incubated in 5% CO2 and 100% humidified air (18 hours). Following the incubation period, embryos were recovered, rinsed, and graded and staged again. The numerical change in the embryo grade (1 through 4) and stage (3 through 8) from the pre-freeze information on the straw label, the post-thaw and post-incubation evaluation were recorded and analyzed with ANOVA.
Results:
Statistically, there was no difference between the pre-freeze, post-thaw, and post-incubation grades or stages between the holding media and PBS+FBS group; however, there was a decrease in the quality grade (P<0.001) following incubation in all groups (Figure 1). The decline in the quality grade following incubation in the holding media and PBS+FBS groups was significantly lower than the decline in the quality grade in the in vitro culture group (Figure 1). There was also a significant decline in the quality grade associated with the freeze / thaw cycle among all the groups (Figure 1). The developmental stage pre-freeze and post-thaw and post incubation was unaffected in the holding media and PBS+FBS groups; however, in the in vitro culture group, on average embryos developed from the morula to the early blastocyst (P<0.001) stage indicating that on average, viability was maintained. This experiment also indicated that approximately 29% of embryos experience some form of damage to the zona pellucida following the freeze / thaw process.
Conclusion:
Cryopreservation of bovine embryos – although critically important to the embryo transfer industry (Mapletoft, 2013), is detrimental to the quality of the embryo and zona pellucida. With the advent of the direct transfer technology, this decrease in quality is not obvious as embryos are seldom observed post thaw. Practically, incubation of poor quality embryos for some time may be a mechanism to salvage a few embryos that have not reached the developmental stage of other embryos in the collection and are normally discarded. The in vitro culture media and system provided a substantially more effective environment to enable embryos to develop further, although the quality of those embryos was negatively affected. It is difficult to determine if the damaging effects of the freeze / thaw cycle can be overcome during an incubation period; however, the damage was apparently suppressed using an in vitro culture approach. Holding media and PBS+FBS while useful as a temporary storage device for bovine embryos is not an adequate short-term incubation media and apparently did not mitigate any damage due to the freeze / thaw process. Future research will involve short-term incubation of fresh embryos in order to eliminate the negative effects associated with cryopreservation. In conclusion, these results and future research may be useful in the bovine embryo industry, and for cattle producers alike, by increasing the number of transferable quality embryos that would otherwise be discarded.
a Megan Bollman, b Ashley Greenhawk, b Ann Shipley, a Philippa Gibbons, a John Gibbons
a College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN 37752,b Hickory Corner Dairy, Speedwell, TN 37870
Introduction:
In the dairy reproduction industry, determining the precise timing for artificial insemination (AI) is a crucial component in obtaining a successful pregnancy outcome. The detection of estrus in dairy cattle is typically characterized by visible behavioral signs such as increased activity and vocalization, aggressive behavior, mounting, and standing to be mounted (Reith & Hoy, 2018). Recognition of estrus has historically been difficult due to behavioral variability among individual animals and environment. The appearance and duration of estrus can be influenced by high milk yield, inadequate nutrition, stress, and overall welfare of an individual animal (Nowicki, Baraniski, Baryczka, & Janowski, 2017). It is also time consuming and expensive for farm staff to monitor the herd for these behavioral signs. Recognition of estrus still remains low even though reproduction management technology strategies, i.e. pressure sensing systems, video cameras, activity meters; have been implemented to ease the task of visually identifying estrus in dairy cattle, (Reith, & Hoy, 2018). Previous studies have shown that a range of 50% of cattle in estrus exhibiting behavioral signs were identified with visual observation to 70% of cattle in estrus were identified using an activity monitoring system (Carvalho et al., 2014). Without the proper technology or technique for estrus detection, strategies to adequately time artificial insemination continue to be a challenge to the dairy industry.
Newer technologies such as timed artificial insemination has been widely used following the synchronization of ovulation in dairy cattle (Wiltbank, & Pursley, 2014). Ovulation synchronization eliminates the need to recognize estrus prior to artificial insemination. Since its introduction in 1995 Ovsynch® and its newer modifications, Presynch-Ovsynch® and Double-Ovsynch®, have almost replaced estrus detection in many dairy herds (Carvalho et al., 2014). By manipulating hormones in order to synchronize ovulation, the challenges of visual estrus identification are reduced, and the number of dairy cattle serviced through timed artificial insemination is increased (Nowicki, Baraniski, Baryczka, & Janowski, 2017). Ovsynch® and its modified protocols may be useful to improve reproduction performance in dairy cattle as it facilitates by appointment breeding and some dairy cows that showed no signs of estrus will indeed be serviced and become pregnant.
The focus of this case study was to evaluate the hormonal response of a dairy herd by observing their ovarian structures following a modified Ovsynch® protocol. The ovarian structures were observed on the day of insemination and retrospectively correlated to pregnancy outcome.
Methods:
A modified Ovsynch® protocol was implemented at a large (≈ 700 cows) local dairy, and is illustrated in Figure 1. Data was collected from lactating dairy cattle from November 28th, 2018 through May 24th, 2019.
On the day of AI, transrectal ultrasonography was conducted to observe ovarian structures. Follicular and corpora lutea (CL) structures were visualized measured and data recorded to retrospectively relate ovarian structures with the pregnancy status on Day 35 post AI. Insemination was conducted regardless of ovarian status, by a single technician using commercially available frozen semen. On Day 35 after AI, transrectal ultrasonography was again used to observe the presence of uterine fluid, ovarian structures, abnormal findings, and to detect the presence of a viable fetus
Results and Discussion:
A total of 60 out of 148 lactating dairy cattle that were analyzed successfully became pregnant following a modified Ovsynch® protocol, giving an overall pregnancy rate of 40.5 ± 0.04% (Table 1). However, the diameter of the largest follicle was not significantly different (P>0.05) between those cows that became pregnant (18.0 ± 0.6mm), and those that did not become pregnant (18.1 ± 0.5mm). Putative cystic cows (largest follicle > 30 mm) were excluded from this analysis; however, 3 of the 6 cows considered to be cystic but were inseminated became pregnant (Largest follicle diameter = 35.3 ± 0.9 versus 40.0 ± 5.0 mm, pregnant versus open). The presence of CL structures in cattle that became pregnant and cattle that did not become pregnant was similar (P>0.05; 25.0 and 29.5%, respectively; Table 1). The diameter of the CL was also similar (P>0.05) between those cows that became pregnant, and those that did not (17.3 ± 1.3 and 16.7 ± 1.0mm, respectively; Table 1).
The average diameters of the ovarian structures (Follicles =18mm, CL =17mm) in lactating dairy cows that became pregnant verse those that did not were further investigated. In a higher (P=0.056) percentage of pregnant cows, the diameter of the largest follicle was ≤18mm (65.0 ± 0.1%) compared to those cows in which the largest follicle was >18mm (35.0 ± 0.1%; Table 2). Although numerically superior, there was no statistical difference in the percentage of pregnant cows with a CL diameter of <17mm (60.0 ± 0.2%) compared to those with a CL >17mm (40.0 ± 0.1%; Table 2).
There was a trend (P=0.131) for a higher percentage of the non-pregnant cows to have a diameter of the largest follicle ≤18mm (55.7 ± 0.1%) compared to those in which the largest follicle >18mm (44.3 ± 0.1%; Table 2). There was no statistical difference in the percentage of non-pregnant cows with a CL diameter of <17mm (52.0 ± 0.1%) compared to those with a CL >17mm (48.0 ± 0.1%; Table 2).
Conclusion:
The use of timed artificial insemination programs and transrectal ultrasonography are beneficial in reproduction management strategies (Colazo, & Mapletoft, 2014). Ovulation synchronization in lactating dairy cows has continued to be an efficient management tool in the dairy reproduction industry. The analysis of ovarian structures following a modified Ovsynch® protocol was useful but not absolute in predicting which cows would become pregnant and which would not. This study determined that although the diameter of the largest follicle in dairy cows at AI did not influence pregnancy rate, a higher percentage of cows that became pregnant had smaller follicles (≤18mm). Any effects of the presence or diameter of the CL on pregnancy status was apparently outweighed by other factors. Further, it is unclear whether the cows that did not become pregnant failed to respond to the synchronization process or were influenced by these factors (nutrition, stress, lactational status, body condition, genetics, etc.). In addition to evaluating overall reproductive health, trans-rectal ultrasonography may be a useful tool for predicting pregnancy outcome. Further research is required to evaluate a more robust ovarian classification system or to evaluate of the endocrine status at the time of AI may also be useful to determine which dairy cows will likely become pregnant or not following Ovsynch® and AI.
Acknowledgements:
The Authors appreciate the assistance and access to the lactating dairy cows provided by Hickory Corner Dairy, Speedwell, TN
References:
Carvalho, P.D., Guenther, J.N., Fuenzalida, M.J., Amundson, M.C., Wiltbank, M.C., Fricke, P.M. (2014). Presynchronization using a modified Ovsynch protocol or a single gonadotropin-releasing hormone injection 7 d before an Ovsynch-56 protocol for submission of lactating dairy cows to first timed artificial insemination. Journal of Dairy Science. 97(10), 6305-6315. Retrieved from: https://www.sciencedirect.com/science/article/pii/S0022030214005244#bib0210
Colazo, Marcos G., & Mapletoft, Teuben J. (2014). A review of current timed-AI (TAI) programs for beet and dairy cattle. The Canadian Veterinary Journal. 55(8), 772-780. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4095965/
Nowicki, A., Baraniski, W., Baryczka, A., & Janowski, T. (2017). Ovsynch protocol and its modifications in the reproduction management of dairy cattle herds-an update. Journal of Veterinary Research. 61(3), 329-336. Retrieved from https://content.sciendo.com/view/journals/jvetres/61/3/article-p329.xml
Reith, S., & Hoy, S. (2018). Review: Behavioral signs of Estrus and the Potential of Fully Automated systems for Detection of Estrus in Dairy Cattle. NCBI 12(2), 398-407. Retrieved from https://pubmed.ncbi.nlm.nih.gov/28807076/
Enrique Gomez a, *, Susana Carrocera a, David Martín a, Juan Jose Perez-Janez b, Javier Prendes b, Jose Manuel Prendes b, Alejandro Vazquez c, Antonio Murillo a, 1, Isabel Gimeno a, Marta Munoz a a Centro de Biotecnologia Animal-SERIDA, Camino de Rioseco 1225, Gijon, 33394, Spain, b Cooperativa de Agricultores y Usuarios de Gijon, Carretera Carbonera 2230, Poligono Industrial de Roces 5, Gijon, 33211, Spain, c Asturian Biotechnology, Galeno, 2248, Poligono Industrial de Roces 5, Gijon, 33211, Spain
Abstract
Direct transfer (DT) of cryopreserved embryos to recipients facilitates on-farm application.We analyzed a new freezing/thawing (F/T) procedure for in vitro produced (IVP) embryos, integrating: 1) an ethyleneglycol based system; 2) a culture step without protein; and 3) a synthetic protein substitute (CRYO3) in cryopreservation medium. IVP embryos from abattoir ovaries were cultured in groups in BSAcontaining synthetic oviduct fluid with or without 0.1% fetal calf serum (FCS) until Day-6. Morulae and early blastocysts were subsequently cultured without protein from Day-6 onwards. Day 7 and Day 8 expanded blastocysts (EXB) were subjected to F/T or vitrification/warming (V/W). Thawed and warmed EXB were cultured in vitro, and development rates, cell counts and dead cells were analyzed in surviving embryos. V/W improved survival over F/T (live and hatching rates at 2 h, 24 h and 48 h) (P < 0.0001), and FCS before Day 6 did not affect in vitro survival. After F/T, embryos had lower cell counts in the ICM, TE and total cells than after V/W. Day-7 embryos after F/T showed % apoptotic, % pycnotic and % total dead cells higher (p < 0.05) than their Day-8 counterparts, probably because F/T reduced the numbers of ICM cells within Day-8 embryos. Thereafter, Day-7 blastocysts were transferred to heifers in an experimental herd. There were no differences in birth rates with frozen (-FCS [n ¼ 40]: 45%; þFCS [n ¼ 14]: 28%), vitrified (-FCS [n ¼ 47]: 53%; þFCS [n ¼ 11]: 36%) and fresh (-FCS [n ¼ 30]: 47%; þFCS [n ¼ 17]: 53%) embryos. However, frozen embryos produced with FCS showed 5/9 miscarriages after Day-40. Calves born from frozen (n ¼ 22), vitrified (n ¼ 29) and fresh (n ¼ 22) transfers did not differ in birth weight, gestation length and daily gain weight (P > 0.10). Subsequently, transfer of frozen embryos (n ¼ 29) derived from oocytes collected from live, hormonally stimulated cows in experimental herd, led to pregnancy rates of 57% (heifers) and 40% (dry cows). with EXB on Day-62 Finally, embryos produced with BSA were transferred to cows in an on-field trial (frozen [n ¼ 80]; fresh [n ¼ 58]), with no differences in pregnancy rates (days 30e40). Pregnancy and birth rates could not be predicted from in vitro approaches. The new F/T system yields pregnancy and birth rates comparable to vitrified and fresh embryos without birth overweight. The absence of products of animal origin, defined chemical composition, and direct transfer entail sanitary, manufacturing and application advantages.
1Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil. Electronic address: gpugliesi@usp.br.
2Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil.
3Department of Veterinary Medicine, School of Animal Science and Food Engineering, University of São Paulo, Pirassununga, São Paulo, Brazil; Embryo SYS, Ouro Fino, MG, Brazil.
5Department of Veterinary Medicine, School of Animal Science and Food Engineering, University of São Paulo, Pirassununga, São Paulo, Brazil.
6Department of Animal Sciences, University of Florida, Gainesville, FL, USA.
Abstract
We aimed to study the association between CL characteristics assessed by color-Doppler ultrasonography (Doppler-US) at the time of embryo transfer (ET) and pregnancy rate (P/ET) in beef recipients. Estrous cycles of crossbred beef recipients were synchronized for timed-ET. On the day of ET (Day 7), CL area, proportion of luteal blood perfusion (BP), and the relationship between the largest dominant follicle (DF) and CL (ipsilateral or contralateral) were determined. Animals (n = 444) received an in vitro produced embryo from Nelore donors, placed in the uterine horn ipsilateral to the CL. Recipients were split retrospectively in three subgroups according to CL area [small (<3 cm2), medium (3-4 cm2), or large (>4 cm2)] and three subgroups according to luteal signals of BP [low (≤40%), medium (45-50%) or high (≥55%)]. Pregnancy was detected on Days 30-45 by transrectal ultrasonography and P/ET was analyzed considering the effects of cow’s category (suckling or non-suckling), CL area, luteal BP and side of DF. P/ET increased along with BP category [low, 45.9%B, (62/135); medium, 54.1%AB (93/172); and high, 58.4%A (80/137)]. When luteal BP was evaluated as a continuous variable, a significant (P < 0.05) linear and positive effect was observed on P/ET. A greater (P < 0.05) CL area and serum progesterone concentrations were observed in the medium and high BP than in the low BP category. Although an effect of luteal size category was not significant on P/ET [small, 49% (76/155); medium, 59.7% (83/139); and large, 50.7% (75/148); P > 0.1], when CL area was evaluated as a continuous variable, a quadratic effect (P < 0.05) indicated a positive relationship between P/ET and CL area until luteal tissue reached 4.07 cm2, followed by a negative relationship. The location of the first-wave DF in relation to the CL did not affect P/ET (P > 0.1). In conclusion, Doppler-US is an innovative tool that has the potential to be used for selection of suitable embryo recipients based on luteal BP. Selection of recipients that have a greater chance of maintaining pregnancy will increase the success of timed-ET programs.
Keywords: Blood perfusion; Cattle; Corpus luteum; Follicle; Pregnancy success.
Gilson Antonio Pessoa 1, Mara Iolanda Batistella Rubin 2, Carlos Antonio Mondino Silva 2, Denize Costa da Rosa 3
1Doutorando do Programa de Pós-Graduação em Medicina Animal-Equinos, Universidade Federal do Rio Grande do Sul, Porto Alegre – RS, Brasil, 2Departamento de Clínica de Grandes Animais, Universidade Federal de Santa Maria, Santa Maria – RS, Brasil, 3Faculdade de Medicina Veterinária, Universidade de Ijuí, Ijuí – RS, Brasil
ABSTRACT
The objective of this study was to evaluate the efficacy of cleaning and decontamination procedures in liquid nitrogen tanks. We evaluated 151 canisters and 133 bottoms from 133 nitrogen tanks of companies or farms for the presence of bacteria and fungi. Samples were collected from the canisters and the bottom of tanks containing liquid nitrogen. Tanks were divided into Group 1 (G1): tanks decontaminated with 2% glutaraldehyde – Glutaron® II (n = 16 canisters in 8 tanks); Group 2 (G2): decontamination with 70% ethanol (n = 20 canisters in 10 tanks); and Group 3 (G3): decontamination with 70% ethanol (n = 115 canisters in 115 tanks). Tanks in Groups 1 and 2 belonged to companies; Group 3 tanks belonged to farms. The culture of canisters showed twelve genera of bacteria and five genera of fungi. Bacillus cereus was the most prevalent bacterial contaminant (42/133) in liquid nitrogen tanks (31.57%). Decontamination by 2% glutaraldehyde plus 70% ethanol was effective and no difference was found between the decontamination methods of Groups 1 and 2. In Group 3 the decontamination method was considered effective. Handling procedures with high hygienic standards should be recommended to avoid contamination of liquid nitrogen tanks on farms.
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