AETA President’s Report – Spring 2020

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Published on: April 6, 2020

The Covid-19 pandemic has most likely altered all of our lives. The AETA board offers our thoughts and support to everyone in this difficult time. The sentiment has been shared that “We are in this together,” and that is certainly true. The AETA and CETA will continue to assess the Covid-19 situation as it evolves and will adjust any plans as needed. The health and safety of our members is our primary concern.

I would like to express my gratitude to Dr. Matt Iager for his service on the AETA board as president. Those of you who have met him or had the privilege to work with him have no doubt witnessed his passion and enthusiasm for the AETA and our profession. I would also like to welcome the newest board members, Dr. Greg Schueller and Dr. Brad Lindsey.

We recently held our spring board meeting and have some great ideas for the future of AETA and AETA related events. Watch the AETA website and the AETA Facebook page for updates and announcements.

To those of you who completed the member survey, thank you! Those who did not, but who want to contribute your thoughts, please seek out a board member. We cannot functionally lead the AETA without member input.

The Convention Committee has been very busy setting the schedule and scope of the upcoming convention. There is something for everyone. We have been releasing information about the program and will continue to do so. Watch the AETA website for updates. Please mark your calendars for October 5–7, 2020, at the Madison Marriott West in Madison, Wisconsin. We hope you can come for World Dairy Expo and stay for the convention.

Once again, we are in this together, and we will come together again soon. I look forward to that!

Thank you,
Matthew Dorshorst, MS, DVM

AASRP AETA Sheep and Goat Seminar

Categories: Catching Up
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Published on: April 6, 2020

The decision has been made to cancel the planned AASRP-AETA Sheep and Goat Embryo Transfer seminar which was planned for June 2020. The faculty have determined that with the current COVID-19 pandemic, the shut-down of Ohio State meetings and courses until at least July1, and the time and planning needed to put together the seminar, it is not possible to reschedule it for 2020. We hope to offer the seminar again in the future.

K. Fred Gingrich II, DVM
Executive Director
American Association of Small Ruminant Practitioners
1130 E. Main St., Suite 302
Ashland, OH 44805
419-496-0696 (office)
419-606-3558 (mobile)
fred@aasrp.org

Practice Tips

Categories: Catching Up, Practice Tips
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Published on: April 6, 2020

Pat Comyn, DVM

1. Always try to obtain straws used in the breeding for a flush to obtain collection date. If the semen is CSS and you’re certified and APHIS inspected, an export opportunity might arise in the future.

2. If a straw label has small print and difficult to read, take a picture and enlarge image.

3. I’ve found that doing procedures, like performing OPU where one really needs an animal to stay still, is greatly eased by administration of 10 mg xylazine with 100 mg ketamine intravenously. This also helps (along with epidural) relieve straining and other things that cows will do while one is attempting a complicated procedure. I prefer doing this as opposed to giving xylazine mixed with a lidocaine epidural; the ketamine seems to provide a more dependable analgesic and sedative effect.

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

Categories: Evidence-Based ET
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Published on: April 6, 2020

John Gibbons, PhD

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.

References:

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.

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

Introduction
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).

Conclusion
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.

References

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.

References:
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

Articles of Interest

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Published on: April 6, 2020

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3211119/

https://www.annualreviews.org/doi/10.1146/annurev-animal-021419-084010

https://www.animal-reproduction.org/article/5b5a6048f7783717068b468e

https://rep.bioscientifica.com/view/journals/rep/156/1/REP-18-0008.xml

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