Effect of Supplemental Trace Minerals on Standard and Novel Measures of Bull Fertility

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Published on: November 2, 2020

T. W. Geary – a2, R. C. Waterman – a, M. L. Van Emon – b, C. R. Ratzburg – c, S. Lake – c, B. A. Eik – a, D. R. Armstrong – a, A. L. Zezeski – a, and J. S. Heldt – d

aUSDA-ARS, Fort Keogh Livestock and Range Research Laboratory, Miles City, MT 59301; bDepartment of Animal and Range Sciences, Montana State University, Bozeman, MT 59717; cDepartment of Animal Sciences, University of Wyoming, Laramie, WY; dMicronutrients USA LLC, 2601 Fortune Circle Drive E. Suite 200C, Indianapolis, IN 46241


Two studies were conducted to evaluate the effects of trace mineral supplementation on traditional and novel measures of bull fertility. In experiment 1, 37 mature bulls received one of three dietary supplements daily for 71 d: 1) Supplement without Cu, Zn, and Mn (CON); 2) Supplement with Cu, Zn, and Mn sulfate (SULF); and 3) Supplement with basic Cu chloride, and Zn and Mn hydroxychloride (CHLR). In experiment 2, 128 Angus or Angus-Hereford calves were maintained on a growing diet for 75 d (year 1) or 119 d (year 2) in Calan gate equipped pens without mineral supplementation. Bulls (n = 32 head/treatment) received one of four trace mineral supplements daily for 84 d: 1) Zn with no Cu (ZN), 2) Cu with no Zn (CU), 3) Cu and Zn (ZNCU), or 4) no Cu or Zn (CON). Fertility measures included a breeding soundness examination (BSE) and novel fertility measures conducted using flow cytometry. In mature bulls, final liver Zn concentration was positively correlated (P = 0.02) with sperm concentration (r = 0.31) and tended (P = 0.06) to be negatively correlated with acrosome damage (r = -0.39). Peripubertal bulls receiving ZNCU had greater ADG than CU bulls (P = 0.05). Each BSE and novel fertility component improved from d 0 to 84 in peripubertal bulls and were not affected (P > 0.10) by mineral supplementation. Bulls that received no supplement (CON) had greater (P < 0.01) percentage of sperm with distal midpiece reflex and Dag defect in their ejaculates. Sperm viability after 30 min of incubation were not affected by trace mineral supplementation, but after 3 h incubation, sperm viability tended to differ (P = 0.06) between treatments and tended to be less for CON bulls compared to ZNCU bulls. Among contrast comparisons, trace mineral supplemented bulls had greater (P < 0.05) percentage of viable sperm at 3 h post collection and reactive oxygen resistant sperm than CON bulls. Addition of Zn to trace mineral containing Cu (ZNCU) improved (P < 0.05) percentage of sperm in the ejaculate with high mitochondrial energy potential and viable sperm with intact acrosome membrane. In summary, it appears the homeostasis mechanisms for bull trace mineral maintenance are extremely efficient and mineral supplementation of mature and peripubertal bulls did not have major improvements in any laboratory or chute-side measures of bull fertility, however bulls exposed to breeding or in environments with diet antagonists might respond differently.

Out of Season Artificial Insemination and Embryo Transfer Results in Ewes: A Field Trial

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Published on: November 2, 2020

B Price, T Mittleider, S Collins, P Gibbons, and J Gibbons

College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN


Ewes are seasonally polyestrous short-day breeders with an estrous cycle of approximately 16 – 17 days.  In the northern hemisphere ewes have active estrous cycles and are naturally receptive to rams from late September to late December when there is less than 12 hours of daylength.  However, progressive sheep breeders often prefer to breed sheep earlier in the year, during periods where there is more than twelve hours of daylength, (July and August) in order to have lambs that are appropriate to target specific show markets.  In order to facilitate this out of season breeding and accelerate genetic gain, producers rely on Assisted Reproductive Techniques such as Laparoscopic Artificial Insemination (LAI), ovarian hyper-stimulation, embryo collection from valuable embryo donors, and embryo transfer (ET) into synchronized recipients (1,2,3.)  This field trial was conducted during late July through early August in southwest Virginia (latitude 36-38’12” N), during a daylight period of about 14 hours. Pregnancy rates of ewes bred by means of AI were compared to those that underwent ET.

7 & 7 Synch: An Estrus Synchronization Protocol for Postpartum Beef Cows

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Published on: November 2, 2020


Researchers at the University of Missouri recently
evaluated a new protocol for synchronization of estrus
among postpartum beef cows. This protocol was found
to be highly effective both for cows receiving embryo
transfer (ET) and cows receiving fixed-time artificial
insemination (AI). Extensive field trials with 7 & 7
Synch observed improvements in the proportion of
cows expressing estrus and in the proportion of cows
becoming pregnant to embryo transfer or to AI.

How can we improve embryo production and pregnancy outcomes of Holstein embryos produced in vitro? (12 years of practical results at a California dairy farm)

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Published on: November 2, 2020

Daniela Garcia Borges Demetrio, 1 ,* Eduardo Benedetti, 2 Clarice Garcia Borges Demetrio, 3 Julio Fonseca, 1 Mayara Oliveira, 1 Alvaro Magalhaes, 1 and Ricarda Maria dos Santos 4

1RuAnn Genetics, Riverdale, CA, United States
2Arizona Dairy Co, Mesa, AZ, United States
3Escola Superior de Agricultura “Luiz de Queiroz”, Universidade do Estado de São Paulo, Piracicaba, SP, Brasil
4Faculdade de Medicina Veterinária, Universidade Federal de Uberlândia, Uberlândia, MG, Brasil


Genomic evaluations have revolutionized dairy cattle breeding, and the demand for embryos produced from very young heifers with high genetic merit has increased over time. The combination of low oocyte recovery, young age of donors, and milk production status can make the in vitro embryo production (IVP) of Holstein cattle incredibly challenging. Several factors need to be coordinated to obtain a live calf from an IVP embryo, but the quality of the oocyte at the start of the process is one of the key factors. Aspects related to oocyte quality, laboratory quality control, embryo quality and recipient selection are addressed here, based on the measures that the RuAnn Genetics Laboratory (Riverdale, California, USA) adopted in the last 12 years, with the goal of improving production of live, healthy calves from Holstein embryos. Follicular wave synchronization and stimulation with follicular stimulating hormone (FSH) is necessary to improve oocyte quality and consequently embryo production. Laboratory quality control and the use of high-quality supplies are essential to reduce variability in production and facilitate identification of other factors that might interfere with embryo production. High pregnancy rates can be achieved with good quality embryos selected at optimal time and stage of development, transferred by an experienced embryo transfer (ET) technician, to well managed recipients 7 or 8 days after estrus. Attention to detail at every step of the process is crucial to success.
Keywords: embryo, Holstein, in vitro production, pregnancy, recipient.

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One-step automated bioprinting-based method for cumulus-oocyte complex microencapsulation for 3D in vitro maturation

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Published on: November 2, 2020

Antonella Mastrorocco1¤a, Ludovica Cacopardo2, Nicola Antonio Martino1¤b, Diana Fanelli3, Francesco Camillo3, Elena Ciani1, Bernard A. J. Roelen4, Arti Ahluwalia2,5☯, Maria Elena Dell’Aquila1☯

1 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, Bari, Italy; 2 Research Centre E. Piaggio, University of Pisa, Pisa, Italy; 3 Department of Veterinary Sciences, University of Pisa, Pisa, Italy; 4 Department of Clinical Sciences, Embryology, Anatomy and Physiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; 5 Department of Information Engineering, University of Pisa, Pisa, Italy

☯ These authors contributed equally to this work.
¤a Current address: Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy
¤b Current address: Department of Veterinary Sciences, University of Torino, Torino, Italy


Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM efficiency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its structural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited. In this study, a bioprinting based production process for generating microbeads containing a COC (COC-microbeads) was optimized and its validity tested in a large animal model (sheep). Alginate microbeads were produced and characterized for size, shape and stability under culture conditions. COC encapsulation had high efficiency and reproducibility and cumulus integrity was preserved. COC-microbeads underwent IVM, with COCs cultured in standard 2D IVM as controls. After IVM, oocytes were analyzed for nuclear chromatin configuration, bioenergetic/oxidative status and transcriptional activity of genes biomarker of mitochondrial activity (TFAMATP6ATP8) and oocyte developmental competence (KHDC3NLRP5OOEP and TLE6). The 3D system supported oocyte nuclear maturation more efficiently than the 2D control (P<0.05). Ooplasmic mitochondrial activity and reactive oxygen species (ROS) generation ability were increased (P<0.05). Up-regulation of TFAMATP6 and ATP8 and down-regulation of KHDC3NLRP5 expression were observed in 3D IVM. In conclusion, the new bioprinting method for producing COC-microbeads has high reproducibility and efficiency. Moreover, 3D IVM improves oocyte nuclear maturation and relevant parameters of oocyte cytoplasmic maturation and could be used for clinical and toxicological applications.

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Sperm DNA Integrity and Male Fertility in Farm Animals: A Review

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Published on: November 2, 2020

Arumugam Kumaresan1*, Mohua Das Gupta1, Tirtha Kumar Datta2 and Jane M. Morrell3

  • 1Theriogenology Laboratory, Southern Regional Station of National Dairy Research Institute (ICAR), Bengaluru, India
  • 2Animal Genomics Laboratory, National Dairy Research Institute (ICAR), Karnal, India
  • 3Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden

The accurate prediction of male fertility is of major economic importance in the animal breeding industry. However, the results of conventional semen analysis do not always correlate with field fertility outcomes. There is evidence to indicate that mammalian fertilization and subsequent embryo development depend, in part, on the inherent integrity of the sperm DNA. Understanding the complex packaging of mammalian sperm chromatin and assessment of DNA integrity could potentially provide a benchmark in clinical infertility. In the era of assisted reproduction, especially when in-vitro fertilization or gamete intrafallopian transfer or intracytoplasmic sperm injection is used, assessment of sperm DNA integrity is important because spermatozoa are not subjected to the selection process occurring naturally in the female reproductive tract. Although sperm DNA integrity testing measures a significant biological parameter, its precise role in the infertility evaluation in farm animals remains unclear. In this review, the earlier findings on sperm DNA integrity in relation to male fertility are compiled and analyzed. Furthermore, the causes and consequences of sperm DNA damage are described, together with a review of advances in methods for detection of sperm DNA damage, and the prognostic value of sperm DNA quality on male fertility.

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Short term incubation of frozen/thawed bovine embryos

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Published on: August 13, 2020

Dalena Hobbs, Colton Holcomb, and John Gibbons, College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN, 37752


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.


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.


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.


AETA Statistics Committee Report – 2010

AETA Statistics Committee Report – 2017

AETA Statistics Committee Report – 2019

Mapletoft, R. (2013, September). History and Perspectives on Bovine Embryo Transfer. Retrieved June 22, 2020, from https://www.animal-reproduction.org/article/5b5a6048f7783717068b468e/pdf/animreprod-10-3-168.pdf

Stadheim, J. A. (2015, April 10). TEST TUBE TECHNOLOGY: Using IVF on cows has numerous pros, cons. Retrieved June 22, 2020, from https://www.tsln.com/news/test-tube-technology-using-ivf-on-cows-has-numerous-pros-cons/

Troxel, T. R. (2013, February 11). Embryo Transfer in Cattle [Scholarly project]. Retrieved June 24, 2020, from https://www.uaex.edu/publications/PDF/FSA-3119.pdf

Ovarian profile and pregnancy rates following ovulation synchronization and timed-artificial insemination in dairy cows

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Published on: August 13, 2020

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


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.


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).


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.


The Authors appreciate the assistance and access to the lactating dairy cows provided by Hickory Corner Dairy, Speedwell, TN


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/

Wiltbank, Milo C., & Pursley, Richard J. (2014). The cow as an induced ovulatory: Timed AI after synchronization of ovulation. Theriogenology 81(1), 170-185. Retrieved from https://www.sciencedirect.com/science/article/pii/S0093691X13003828

Efficient one-step direct transfer to recipients of thawed bovine embryos cultured in vitro and frozen in chemically defined medium

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Published on: August 13, 2020

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


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.

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Use of color-Doppler ultrasonography for selection of recipients in timed-embryo transfer programs in beef cattle

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Published on: August 13, 2020

Guilherme Pugliesi 1Gabriela Dalmaso de Melo 2Júlio Barboza Silva 3Alexandre Sardinha Carvalhêdo 4Everton Lopes 2Emivaldo de Siqueira Filho 4Luciano Andrade Silva 5Mario Binelli 6

  • 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.
  • 4Embriotec Reprodução Animal, Anápolis, GO, 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.


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.

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Decontamination of naturally contaminated liquid nitrogen storage tanks

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Published on: August 13, 2020

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


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.

Key words: artificial insemination; bacteria; fungi; semen

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The effects of zinc on the maturation and fertilization of bovine oocytes

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Published on: April 6, 2020

Brianna M. Price, Taylor F. Mittleider, Kayla Grau, and John Gibbons,
College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN

Zinc is an essential trace mineral in many species, playing many roles including an essential role in reproduction. Zinc is found throughout the body including the brain, kidney, liver, muscle, and bones where it plays a role in RNA and DNA metabolism (Hambridge and Krebs, 2007.) The highest concentrations of zinc are found in the eye and prostate gland (Hambridge and Krebs, 2007.) In the bovine oocyte, zinc is the most abundant transition metal with concentration fluctuations occurring during maturation and fertilization events (Que et al., 2019.) In all organisms examined, an event referred to as the “zinc spark” has been documented as an essential reproductive phenomena (Que et al., 2019.) High concentrations of zinc are present in the female gamete prior to the zinc spark, where zinc is released from the oocyte following intracytoplasmic sperm injection and natural encounters with a sperm cell (Bernhardt et al., 2012; Duncan et al., 2016; Que et al., 2019.) Higher quantities of zinc release during this event has been associated with higher quality embryos (Duncan et al., 2016; Picco et al., 2010; Zhang et al., 2016.) Zinc has been shown to play an important role in DNA stabilization during the fertilization process, when DNA is in a haploid state, including protection from damage and apoptosis (Anchordoquy et al., 2014.) The ability to produce in vitro bovine embryos provides an ideal model to enhance understanding of fertilization events in human reproduction. This studied examined the role of zinc in in vitro maturation and fertilization of bovine oocytes and tested the hypotheses that dose dependent zinc supplementation would enhance oocyte maturation and the chelation of zinc would inhibit fertilization and early embryonic development.

Evaluation of Zinc Supplementation on In Vitro Maturation
Bovine oocytes were obtained via follicular aspiration of postmortem ovaries harvested from an abattoir. Selected oocytes contained at least three layers of cumulus cells and a homogenous cytoplasm. Oocytes were separated into four in vitro maturation treatment groups supplemented with 0, 5, 10, and, 20 μM zinc. Supplementation doses where determined from analysis of zinc concentrations in adult cow plasma and follicular fluid (10.55 μM and 11.47 μM, respectively) and commercial maturation and fertilization medias (1.07 μM for each). Oocytes were considered mature if they had reached Metaphase II and had expelled their first polar body after 18 hours in maturation media. There was no statistical significance found in the maturation rates of the oocytes (78.1 ± 3.0%, 59.5 ± 4.3%, 69.8 ± 7.7%, 62.3 ± 3.2%, respectively). Mature oocytes were statistically analyzed by Chi Square test.

Evaluation of Zinc Chelation on In Vitro Fertilization and Embryo Development
The effects of zinc chelation on fertilization was observed in oocytes matured in 0 μM zinc, fertilized with frozen-thawed bull semen of a characterized bull, and separated into two groups. A zinc chelated group contained 2.7 mM of TPEN (tetrakis(2-pyridinylmethyl)-1-2-ethanediamine) supplemented in the fertilization media compared to non-treated controls. Following fertilization, the presumptive zygotes were cultured in their respective groups for 7 days (no TPEN). Embryonic development to the morula or blastocyst was analyzed by Chi Square test. The TPEN treated group had a statistically lower cleavage rate (p<0.05) than the control group (46.1 ± 2.3% and 75.6 ± 3.4%, respectively). Embryo development rate to morula stage was also statistically lower (p<0.05) in the TPEN treated group compared to the controls (15.4 ± 0.03% and 37.8 ± 0.03%, respectively). The average embryo developmental stage scores analyzed by ANOVA were significantly lower (P<0.001) in the TPEN treated group compared to the controls (2.2 ± 0.1 and 3.4 ± 0.2, respectively).

This study supports the concept that zinc supplementation has minimal effects on in vitro maturation of oocytes; however, removing zinc during in vitro fertilization, significantly decreased cleavage rate and embryo development to blastocyst. Future studies may determine a more precise role of Zinc during sperm penetration and fertilization mechanisms.


Anchordoquy, J. M., Anchordoquy, J. P., Sirini, M. A., Picco, S. J., Peral-García, P., & Furnus, C. C. (2014). The Importance of Having Zinc During In Vitro Maturation of Cattle Cumulus-Oocyte Complex: Role of Cumulus Cells. Reprod Dom Anim, 49, 865-874. doi:10.1111/rda.12385

Bernhardt, M. L., Kong, B. Y., Kim, A. M., O’Halloran, T. V., & Woodruff, T. K. (2012). A Zinc-dependent mechanism regulates meiotic progression in mammalian oocytes. Biology of Reproduction, 86(4):114, 1-10. doi: 10.1095/biolreprod.111.097253

Duncan, F. E., Que, E. L., Zhang, N., Feinberg, E. C., O’Halloran, T. V., & Woodruff, T. K. (2016). The zinc spark is an inorganic signature of human egg activation. Scientific Reports, 6,24737. doi: 10.1038/srep24737

Hambridge, K. M. & Krebs, N. F. (2007). Zinc deficiency: a special challenge. Journal of Nutrition, 137(4), 1101-1105.

Picco, S. J., Anchordoquy, J. M., de Matos, D. G., Anchordoquy, J. P., Seoane, A., Mattioli, G. A., Errecalde, A. L., & Furnus, C. C. (2010). Effect of increasing zinc sulphate concentration during in vitro maturation of bovine oocytes. Theriogenology, 74, 1141-1148. doi:10.1016/j.theriogenology.2010.05.015

Que, E. L., Duncan, F. E., Lee, H. C., Hornick, J. E., Vogt, S., Fissore, R. A., O’Halloran, T. V., & Woodruff, T. K. (2019). Bovine eggs release zinc in response to parthenogenetic and sperm-induced egg activation. Theriogenology, 127, 41-48. doi:10.1016/j.theriogenology.2018.12.031

Zhang, N., Duncan, F. E., Que, E. L., O’Halloran, T. V., & Woodruff, T. K. (2016). The fertilization-induced zinc spark is a novel biomarker of mouse embryo quality and early development. Scientific Reports, 6, 22772. doi:10.1038/srep22772

Dominant follicle removal prior to superovulation

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Published on: April 6, 2020

Taylor Mittleider, a Brianna Price, a John Gibbons, a Jason Anton b
a College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN, b Ovaflo Genetics, Tahlequah, OK

Introduction: Superovulation and embryo collection and transfer enables cattle producers to reach reproductive, financial, and genetic goals. Although knowledge of follicular development has improved, the number of transferable embryos per collection has not, leading to a high degree of unpredictability. Follicular stimulating hormone (FSH) is a major cost of embryo transfer, and administration must occur coincidentally with an endogenous FSH surge for effective superovulation and embryo recovery, which has not improved substantially in many years, possibly due to suboptimal timing of FSH delivery (Adams, 1992). A major source of variability in the superovulatory response in cattle is the status of ovarian follicles at the time of initiation of FSH treatments (Mapletoft, Steward, & Adams, 2002). Following dominant follicle ablation, an FSH surge and associated follicular wave can be predicted and managed, which may lead to more consistent embryo collections and more transferable embryos (Crowe, 2013). The purpose of this field trial was to evaluate dominant follicle ablation prior to superovulation with a minimal dose of FSH.

Methods: Cycling beef cattle, at random stages of the estrous cycle , were subjected to transvaginal ultrasound-guided aspiration of all follicles (> 5 mm). Following aspiration, PGF2a (25 mg) was administered and a CIDR was placed. Approximately 48 hours later, Folltropin-V administration began and was given twice daily (am and pm) for 4 days. On the third day of FSH administration, PGF2a was given again and the CIDRs were removed that evening. Cattle were inseminated at estrus. One week later, embryos were collected and corpora lutea (CL) were counted using transrectal ultrasonography. All data, both pre-recovery and day of recovery, were analyzed statistically using ANOVA.

Results: Neither the number of follicles ablated, nor the diameter of the ablated follicles had any statistical effect on embryo recovery; however, as indicated in Table 1, cattle (n = 24) with a CL < 22 mm at ablation, tended (P = 0.086) to produce fewer transferable quality embryos (mean ± SEM; 5.8 ± 0.7) than cattle (n = 26) with a CL ≥ 22 mm (8.1 ± 1.1) at ablation.

Cattle (n = 35) given ≥ 10 mls of FSH had a similar number of; total ova (11.3 ± 1.3), transferable embryos (6.2 ± 0.9), and CL (14.2 ± 0.9) compared to cattle (n = 28) given < 10 mls of FSH (12.3 ± 1.1, 6.3 ± 0.6, 15.1 ± 1.0, respectively). This approach also facilitated acceptable results from consecutive embryo recoveries (Figure 1).

Conclusion: Dominant follicle removal prior to superovulation, required less exogenous FSH to achieve acceptable embryo recovery results. These results indicated that ablation of follicles (> 5mm) in cycling mid-diestrus beef cattle, prior to initiation of superovulation may yield more consistent embryo production perhaps due to a more tightly synchronized engineered follicular wave. Further characterization of the dynamics of this follicular wave may facilitate more consistent superovulation results and reduce costs.

Adams GP, Matteri RL, Kastelic JP, Ko JC, Ginther OJ. Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers. Journal of Reproduction Fertility, 1992; 94(1):177-188.

Crowe MA, Mullen MP. Relative roles of FSH and LH in stimulation of effective follicular response in cattle. Intech Open Access, 2013; http://dx.doi.org/10.5772/50272.

Mapletoft, R. J., Steward, K. B., & Adams, G. P. (2002). Recent advances in the superovulation in cattle. Reproduction, nutrition, development, 42(6), 601–611. https://doi.org/10.1051/rnd:2002046

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Published on: April 6, 2020





What is an early blastocyst? (And does it matter?)

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Published on: January 3, 2020

Written by Dr. Jennifer Barfield

This year at the annual AETA/CETA meeting in Colorado Springs approximately 100 people signed up for a pre-conference symposium on Advanced ET. One of the three sessions was embryo grading where the audience was asked to stage and grade a variety of embryos from both pictures and videos. This is an exercise that we have done at this meeting in the past and it was interesting to revisit some of the same questions. As in the past, I polled the participants in this session to gauge what level of expertise was in the audience. The breakdown according to years of experience was 0-5 (25%), 6-10 (19%), 11-15 (12%), 16-20 (12%), and 21+ (32%).

For many of the questions the results showed a general consensus, but there was one question for which the distribution was not what I expected. The slide below is the question as it was presented to the audience. The embryo photo is from the IETS Bovine In Vivo Ova Tutorial (see p. 85, IETS members can access the document here https://www.iets.org/pubs_educational.asp).

Across all 3 sessions the answers were A 7%, B 54%, C 34%, D 1%, and E 4% with more disagreement in the first and third session than in the second. See Figure 2. In all sessions more people classified this embryo as an early blastocyst but the number of people who classified this a full blastocyst surprised me. I went back to look at the IETS guide and it stated that as the blastocoele of this embryo is approaching 50% of the embryo that this may be considered a stage 6 embryo.

As someone who often teaches students about classifying and grading embryos, the idea that I may have been instructing people incorrectly troubled me. At Colorado State University, I teach that an early blastocyst is one in which there is a blastocoele cavity present that has not yet filled the perivitelline space of the embryo, even if the blastocoele cavity is larger than 50% of the embryo. A full blastocyst is characterized by a blastocoele cavity that touches the zona pellucida on all sides, except for where it touches the inner cell mass, thus there is no PV space. I wondered if perhaps my interpretation of an early blastocyst is the result of a drift in teaching from an earlier time when these definitions were more strictly and/or widely followed. So I broke down the answers for this question according to years of experience thinking that perhaps practitioners who were learning how to classify embryos when the guidelines were developed would adhere to them more strictly, i.e. more often calling an embryo like this a full blastocyst. That wasn’t the case.

Of the respondents who had over 20 years of experience, 69% classified this embryo as an early blastocyst (24/35) while 52% of the youngest cohort classified this embryo as early (14/27). The only group in which more people called this a blastocyst than an early blastocyst was the 11-15 years group, although there were few respondents overall in this group (8/13 called this a blastocyst). So, I was wrong about the oldest yet wisest of us following the IETS staging guidelines more strictly.

That led me to ask the question, does it even matter if we are all calling this embryo an early blastocyst or a blastocyst? If you are collecting and transferring day 7 in vivo-produced embryos, probably not. The pregnancy rates from transferring grade 1 early blastocysts and grade 1 blastocysts are not significantly different (Hasler, 2001). This slight difference in stage would not change the synchrony of the recipient you choose. It would likely not change how you would cryopreserve this embryo as most in vivo embryos are slow frozen rather than vitrified. From a research standpoint we often make distinctions in stage depending on the question being asked, so it’s possible that inconsistencies in classifying embryos in the field could yield some erroneous conclusions, although I’ll admit I cannot give you any examples of this as I have not scoured the literature for papers where there were significant differences in outcome between early blastocysts and blastocysts for any tested hypothesis.

Outside of simply desiring consistency, the only time when the decision to call it an early blastocyst verses a blastocyst may be important is when grading in vitro-produced embryos. Grading embryos is not only based on the physical appearance of the embryo but also on its stage of development. The slide below was also discussed during the embryo grading sessions in the context of how to incorporate stage into overall embryo grade. In vitro-produced embryos are approximately 1 day more advanced in development than in vivo-produced embryos because of what we consider day 0 in these 2 systems (in vivo day 0 = standing heat, in vitro day 0 = initiation of co-incubation of sperm and oocytes). All morulae would be a day behind in development in an IVP system and given a grade 2 no matter how perfect but early blastocysts sit on the fence. Grading may be an instance where the > or < 50% blastocoele volume distinction matters with embryos with <50% of the volume being the blastocoele cavity being grade 2 and those with >50% volume being blastocoele grade 1. Still, I would be surprised if this fine distinction and difference in grade would translate into a significant difference in pregnancy rates, which is what matters. If anyone has data that may provide insight, please share it! 

So does it matter if we are all calling this small subset of embryos early blastocysts or blastocysts? Probably not, at least not for in-vivo produced embryos. Is it interesting? I think so, particularly from an educational and research perspective. Is it something that we as a community of reproductive practitioners and embryologists should talk more about as we consider developing a separate grading system for in vitro-produced embryos? I’d say yes.

How much Follicle Stimulating Hormone do we really need for cattle superovulation ?

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Published on: January 3, 2020

Superovulation data

Although the American Embryo Transfer Association and the International Embryo Technology Society perform a tremendous and necessary review of embryo transfer activity in the United States (Tables 1 and 2) and worldwide, there are limited data available on the dose, type, route of delivery, and protocols for Follicle Stimulating Hormone (FSH) administration (Kelly, 1997).  Other factors that contribute to the success of ovarian hyperstimulation are the breed, age, parity, and management of cattle, ovarian follicular reserve, and superovulation history of a particular donor.  Delivery of FSH to achieve superovulation is generally a twice daily injection schedule beginning on the day before or the day of emergence of a follicular wave (Adams, 1992) and lasting for three or four days; however, single dose (Looney, 1986; Bo, 1994; Kelly, 1997) or split single dose delivery (Tribulo, 2012), as well as FSH gels (Kimura, 2016) and implants (Floyd, 2007) to enhance bioavailability have been reported.  The current FDA approved FSH product is a pituitary derivative although the interest in producing a custom, reliable, and effective, FSH (and Luteinizing Hormone [LH]) product from recombinant technology has a substantial history (Looney, 1988; Wilson, 1993) and is gaining considerable traction (Hesser, 2011; Vega, 2019).  Classically, pituitary-derived FSH products had substantial LH contamination and a role for each of the gonadotropins was hypothesized (Donaldson, 1985).  The current product is very pure although it is likely that some LH might well be important for successful nourishment of multiple dominant follicles (Ginther, 1996) although it may be difficult to mimic the pulsatile pattern of LH.  Regardless of the protocol, the most critical component for FSH administration is the timing relative to the endogenous FSH surge.  Practically, this approach requires a hormonal or mechanical technique to engineer a follicular wave in order to efficiently schedule the embryo collection (Crowe, 2013.  The protocol for engineering a follicular wave also has many considerations and challenges (time, expensive equipment, choice of hormones, etc.).

What if we miss an FSH injection?

The literature is scant with information about which FSH injections are the most important.  It seems logical that the first few injections are the most important (due to dosage and timing) and the last few are the least important.  Using a six FSH injection protocol following ultrasound guided follicular ablation of all follicles larger than 5 mm, the administration of the sixth FSH injection or not did not impact the embryo recovery results (Gibbons, 2019).  Practically, even if it is known that an FSH injection was missed, the donor will still likely be inseminated and embryo recovery attempted.  A single dose of FSH administered on Day 10 following estrus has been shown to produce a similar number of ovulations as a multi-dose approach (Kelly, 1997); however, there were more degenerate embryos and unfertilized ova, suggesting that in addition the scheduling aspect, engineering a follicular wave for superovulation may be important impact the “fertilizability” of the ova within the follicles and the timing of the first few FSH injections relative to follicular wave emergence outweighs the effects of any other single FSH injection.

FSH per Transferable Embryo

There is no public data base for the amount of FSH given to any one donor.  There are recent data (Gibbons, 2019) to suggest that the amount of FSH per transferable embryo may be as low as 1.5 mls (54 IU; Folltropin) following an engineered follicular wave.  The appropriate timing of FSH initiation could decrease the overall required dosage of FSH, which is financially important given that the cost of FSH is one of the largest single costs associated with superovulation.  Further, although there is a relatively accurate idea of how many corpora lutea (CL) are present at embryo collection, without counting the CL via ultrasonography, it is difficult to know if or how many embryos / ova are not accounted for following collection. 

Where do we go from here?

In vitro embryo technologies are clearly gaining considerable traction (Table 2.); however, the need for effective and efficient superovulation protocols remains important.  The effectiveness of these protocols is linked to the timing of the initial FSH injection; however, due to the considerable number of different protocols that are available it is difficult to determine which approach more appropriately exploits the endogenous FSH surge and results in more transferable embryos.  Future research comparing different FSH protocols relative to endogenous FSH profiles and follicular wave emergence will be important and may increase the number of transferable embryos per collection which has not waivered substantially in 20 plus years.


Adams GP, Matteri RL, Kastelic JP, Ko JC, Ginther OJ.  Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers.  Journal of Reproduction Fertility, 1992; 94(1):177-188.

Bo GA, Hockley DK, Nasser LF, Mapletoft RJ.  Superovulatory response to a single subcutaneous injection of Folltropin-V in beef cattle.  Theriogenology, 1994;42(6):963-975.

Crowe MA, Mullen MP.  Relative roles of FSH and LH in stimulation of effective follicular response in cattle.  Intech Open Access, 2013; http://dx.doi.org/10.5772/50272.

Donaldson LE.  LH and FSH at superovulation and embryo production in the cow.  Theriogenology 1985;23(3):441-447.

Floyd C.  Subcutaneous FSH implants.  MS Thesis, Clemson University, 2007: https://tigerprints.clemosn.edu/all_thesis/94.

Gibbons JR, Anton J.  Dominant follicle removal prior to superovulation.  Poster presented at 2019 joint annual AETA & CETA/ACTE convention, 2019.

Ginther OJ, Wiltbank MC, Fricke PM, Gibbons JR, Kot K.  Selection of the dominant follicle in cattle.  Biology of Reproduction 1996;55:1187-1194.

Hesser MW, Morris JC, Gibbons JR.  Advances in recombinant gonadotropin production for use in bovine superovulation.  Reproduction Domestic Animals, 2011;46:933-942.

Kelly P, Duffy P, Roche JF, Boland MP.  Superovulation in cattle: effect of FSH type and method of administration on follicular growth, ovulatory response and endocrine patterns.  Assisted Reproduction Sciences 1997;46:1-14.

Kimura K.  Superovulation with a single administration of FSH in aluminum hydroxide gel: a novel superovulation method for cattle.  Journal of Reproduction Development, 2016;62(5):423-429.

Looney CR, Bondioli KR, Hill KG, Massey JM.  Superovulation of donor cows with bovine follicle-stimulating hormone (bFSH) produced by recombinant DNA technology.  Theriogenology 1988;29:271.

Looney CR.  Superovulation in beef females.  Proceedings of the 5th annual conference of American Embryo Transfer Association, 1986;16-29.

Tribulo A, Rogan D, Tribulo H, Tribulo R, Mapltoft RJ, Bo GA. 

Superovulation of beef cattle with a split-dose intramuscular administration of Folltropin-V in two concentrations of hyaluronan.  Theriogenology 2012;77:1679-1685.

Vega VMB, Chavez SPJ, Franco CDM, Ramos TI, Toledo JR.  FSH in superovulation.  Revista Bionature, 2019;812-816.

Wilson JM, Jones AL, Moore K, Looney CR, Bondioli KR.  Superovulation of cattle with a recombinant-DNA bovine follicle stimulating hormone.  Animal Reproduction Science, 1993;33(1):71-82.

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Published on: January 3, 2020

The pre-hatching bovine embryo transforms the uterine luminal metabolite composition in vivo

Somatic cell nuclear transfer alters peri-implantation trophoblast differentiation in bovine embryos

Placental development during early pregnancy in sheep: Effects of embryo origin on vascularization

Bovine Fetal Placenta During Pregnancy and the Postpartum Period

Heifer nutrition during early- and mid-pregnancy alters fetal growth trajectory and birth weight

Reduced quality of bovine embryos cultured in media conditioned by exposure to an inflamed endometrium

Pivotal periods for pregnancy loss during the first trimester of gestation in lactating dairy cows

Evaluation of the uterine environment early in pregnancy establishment to characterise cows with a potentially superior ability to support conceptus survival

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

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Published on: July 26, 2019

Read full article here

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



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


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


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.


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


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.


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).


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.


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.


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.



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

In vitro culture systems: how far are we from optimal conditions?

Categories: Research Publications
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Published on: July 25, 2019

C. Wrenzycki


Anim Reprod, vol.13, n3, p.279-282, 2016


Over the past decades in vitro production (IVP) of bovine embryos has been significantly improved. Nevertheless, embryos generated in vitro still differ from their in vivo produced counterparts. Embryos must adjust to multiple microenvironments at preimplantation stages. Consequently, maintaining or mimicking the in vivo situation in vitro will aid to improve the quality and developmental competence of the resulting embryo.

cattle, embryo, in vitro production

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Published on: April 17, 2019

Significant heparin effect on bovine embryo development during sexed in vitro fertilization

Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: Implications for blastocyst yield and blastocyst quality

Sex control by Zfy siRNA in the dairy cattle

Daily administration of a GnRH analogue enhances sperm quality in bucks during the non-breeding season

Maternal age influences the number of primordial follicles in the ovaries of yearling Angus heifers

Role of cAMP modulator supplementations during oocyte in vitro maturation in domestic animals

Factors in cattle affecting embryo transfer pregnancies in recipient animals

Comparison of luteolysis and timed artificial insemination pregnancy rates after administration of PGF2α in the muscle or the ischiorectal fossa in cattle

Large-scale transcriptional analysis of bovine embryo biopsies in relation to pregnancy success after transfer to recipients

Effects of supplementation of medium with different antioxidants during in vitro maturation of bovine oocytes on subsequent embryo production

Influence of bovine serum albumin and fetal bovine serum supplementation during in vitro maturation on lipid and mitochondrial behaviour in oocytes and lipid accumulation in bovine embryos

50 Survival of sexed ivf-derived bovine embryos frozen at different preimplementation stages of development

141 Bovine embryo development rats are affected when oocytes are matured in different vials containing hepes/bicarbonate buffered medium

The ischiorectal fossa: an alternative route for the administration of prostaglandin in cattle

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