Brief Summary of Minutes of Annual Meeting

David Towson. Welcome and room is reserved until 5:00 pm. Lunch will be here and there will be welcoming words from Jon Wraith, Associate Director, NH Agricultural Station.

Joy: requests for 2008 publications to all members of the group. What that USDA want regarding publications? Papers, abstracts, thesis, Station reports. Adele, indicated more is better. Then, it was decided that Papers, thesis and Station reports were all useful. Do not include abstracts.

Joy: Rafael is presently secretary and will move to Chair; Jennifer will move from Director to Secretary and a new director will need to be selected.

Station Reports:

MA-Station-Rafael Fissore-Umass Amherst.

• Bovine PLCzeta can be a very useful tool to test developmental competence, as it initiates sperm-like oscillations in bovine eggs.
• Definition of repeat breeders. A question that needs to be addressed is that oocytes in lactating cows the follicular size may be larger than in heifers, but more importantly they may have much longer exposures to hormones, which may seriously compromise the developmental competence of oocytes.
• This will be very interesting to test whether the oocytes markers decrease, as aging happens in the follicle.

Nebraska Station- Jennifer Wood.

• Interested in examining the effect of metabolism on oocyte development. Using a -/- agouti mutant that overeats as a model. Significant amounts of fat in the carcass.
• These animals become infertile by about 6 months. The notion is that there is not a normal signal for ovulation. The thought is that gonadal fat pad may very important in limiting the reproductive capacity of these animals.
• Leptin levels are much higher in these animals than in the controls, even as early as 12 wks.
• Energy balance may be more critical than the absolute weight. Looking at genes in granulosa cells in these animals, StAR expression was up in the mutant mice while Egr-1 was decreased.
• BXD animals crosses obtained from the progeny of C57Bl and DBAs. They have several 30 lines and will subject them to different diets to see how they respond in terms of oocyte quality and fertility.
• In a cow project, testing the number of antral follicles in culled cows showed that culled cows at later times seem to have more antral follicles.

Erdogan Memili - Mississippi State U

• Effect of nutrition/metabolism, lactation, and immunology on reproduction
• Focus = embryo development/programming; epigenetic changes during development
• Molecular mechanisms of oocyte quality and embryonic developmental competence
• Role of sperm in fertility - differentially expressed proteins/SNPs associated with bull fertility
  • DNA damage associated with low fertility
• Maternal influence - Bovine germinal vesicle and cumulus cell proteomics study - Reproduction
  • DNA methylation/chromatin remodeling of oocyte/cumulus cells
• Embryonic genome activation - chromatin remodeling, DNA methylation, and LAG (leukocyte antigen G; immune function) from 1-cell to 8-cell

Ned Place - Cornell

• Photoperiod and fertility; primordial follicle activation
• Model = Siberian hamsters (long-day breeders); more AMH in SD breeders - suppresses folliculogenesis
• Responsiveness of cows to ovarian stimulation - initial studies in hamsters = model for responsiveness; during short days - poor response/low ovulation rate while during long days - good response/high ovulation
• Some genes were up regulated in the long day animals vs. short when expression was analyzed with microarrays. They got 50% coverage of the gene expression profiles in oocytes. There were 98 genes that were differentially expressed among these genes, one of which was aromatase. Animals exposed to LDays will eventually lose all the ovarian mass prematurely compared to animals that have been raised with the normal amount of light and photoperiod.

Jon Wraith - Associate Director-NH Station.

• Comments. Welcoming remarks. Arrived from Montana State. Happy to be here. Trying to re-invigorate agriculture in NH.

Joy Pate - Penn State

• Smad4 KO in oocyte (ZP3 CRE) or granulosa only - phenotype (Francisco Diaz).
  • These mice have more follicles and oocytes than controls. The same results were obtained with pharmacological inhibitors of this pathway. It seems that Smad4 mutant oocytes are larger, but have fewer granulosa cells. These results suggest that oocytes control granulosa cells proliferation, which is well known finding.
• AMH - prevents follicular maturation due to reduced granulosa proliferation; as follicles grow  lose AMHR resulting in AMH insensitivity (AL Johnson)
• Interaction between luteal cells and T cell lymphocytes; Contact-mediated and contact-independent effects
  • Brefeldin A (BFA) blocks conventional secretion of proteins and increases unconventional secretion of proteins.
  • Luteal cell conditioned medium induces luteal cell dependent proliferation of T cells; if you add BFA too, T cell proliferation is enhanced suggesting an unconventional secreted product that causes the proliferation
  • Gamma-delta T cell proliferate in absence of contact; if you add CD4 or CD8 (alpha-beta) cells with luteal cells, there is poor proliferation of T cells (this is during midcycle)
  • At CL regression, contact between luteal cells and gamma-delta is required for T cell proliferation
• miRNA in luteal cells (day 4 vs day 10 vs d18 (pregnant and non-pregnant))
  • microarrays for miRNA - shows differences
  • knock-down Dicer and Drosha - identifying functional end-points

Paul Tsang - U New Hampshire
Bovine Corpus Luteum Capillary Endothelial Cell Cultures

• 4 subtypes of endothelial cells- what are proportions of these types during corpus luteum development . What is functional importance
• Migration of endothelial cells dependent on MMP production; regulation of MMP-2 by progesterone - high concentration of progesterone inhibits MMP-2 activity; endogenous inhibitor (TIMP1) also decreased upon progesterone treatment. However, TIMP2 levels increased upon progesterone treatment
• MMP2 and MMP9 detected in Penn State CL lysates
• Developed protocol to isolate and culture endothelial cells from CL
• Problems with cryopreservation; primary cultures  decided to investigate culture and cryopreservation properties
• How many passages? - 2 passages, cells look good (appropriate morphology)
• Cryopreservation - DMSO concentration (10% instead of 2.5%)  cells recovered much better.
• Future studies:
  • endothelial cell verification (factor VIII, acetylated LDL, progesterone in conditioned medium) - interestingly, endothelial cell conditioned medium contains detectable progesterone. Probably contaminating luteal cells - how to get rid of these contaminating cells?
  • Comparison studies using bovine adrenal capillary endothelial cells; clonal cultures

Milo Wiltbank - U Wisconsin

• Role of progesterone in fertility
• Pre-AI progesterone
  • In first lactation animals - Double Ovsynch increases fertility dramatically (but not in older animals); produces high progesterone due to production of a second CL - High P4 =higher pregnancy per AI (50% versus 37%); also less pregnancy loss (P = 0.054); less double ovulation (therefore increased twinning) - double ovulations still relatively high
  • Double Ovsynch with extra PGF2a  more complete luteolysis  appx 5% increase in PP/AI-first service (not statistically significant) but not necessarily important for improving fertility
• Post-AI progesterone
  • Change - 5 days after AI add hCG or supplement with CIDR
  • CIDR + hCG in lactating cows raises P4 to match heifers
  • However, there is no effect of this treatment on conception rate suggesting that P4 prior to AI is more important than post-AI (smaller group of animals)
  • In ~3000 lactating cows, hCG did have an effect (37% v 42%) (second study with larger group of numbers)
• Endometrial thickness near timed AI (ultrasound)
  • When P4 is high, endometrial thickness is low (~6.9 mm); as P4 drops precipitously with PGF2a injection, endometrial thickness increases (9.2  9.4 mm)
  • P4 concentration seems to determine endometrial thickness (inversely correlated)
  • Compared groups (endometrial thickness < 8mm versus >8 mm); endometrial thickness was an important indicator for fertility (e.g improved pregnancy /AI) predicts about ½ of the fertility
  • Mechanism is not known??

Ron Butler - Cornell

• Embryo survival in lactating versus non-lactating dairy cows (oocyte quality); embryo transfer to lactating cows vs. virgin Holstein heifers
• Effects of conjugated linoleic acid (CLA) on reproduction
  • CLA-10,12 isoform improves dairy cow fertility (meta-analysis)
  • Possible mechanisms = earlier first ovulation, increased plasma IGF-1, improved estradiol production from the ovarian follicle, no effect on uterine PGF2a production
• Trans (18:1) fatty acids which may increase CLA improved reproduction due to increased fertilization rate and embryo quality
• Developed IVM/IVF protocol
  • Parthenogenesis studies first
  • CLA during IVM only - no statistical differences in MII maturation, cleavage rate, or blastocyst development
  • CLA during whole culture (IVM ) - start to see an effect however they seem to be inhibitory especially in blastocyst rate
  • Next step - repeat above doing IVM/IVF. Also, include in samples from lactating and non-lactating animals and embryo transfers

Brad Hillman

• Appx. 1 year out for re-write; start planning organization of writing; want to begin review process at beginning of 2012.
• Size of group - small; not necessarily a problem unless loose critical mass
• NIFA - 25% of Hatch must go to multi-state - make sure they are true collaborative endeavors-grant writing/manuscripts
• Submit meeting minutes within 60 days (SAE 422??) - Rubie Mize = assistant for Brad
• Emphasize collaborations and importance of these collaborations for re-write and during development of new application

Adele Turzillo
Overview and Update of NIFA programs - see handouts

New Director: Milo Wiltbank - Joy nominates, several individuals second the nomination - passed unanimously

Plan for Re-write
Groups:

• Oocyte/Embryo
• CL
• Metabolism/Lactation/Reproductive Efficiency

Location for 2011: Washington DC - meet at USDA building - Adele would host

2. Araujo RR, Ginther OJ, Ferreira JC, Palhao MM, Beg MA, Wiltbank MC, 2009. Role of follicular estradiol-17² in determining the time of luteolysis in heifers. Biology of Reproduction PMID 19264702. Mar 4.

3. Souza AH, Cunha AP, Silva EPB, Gumen A, Ayres H, Guenther JN, Wiltbank MC, 2009. Comparison of gonadorelin products in lactating dairy cows: efficacy based on induction of ovulation of an accessory follicle and circulating LH profiles. Theriogenology 72:271-279.

4. Brusveen DJ, Souza AH, Wiltbank MC, 2009. Effects of additional prostaglandin F2± and estradiol-17² during Ovsynch in lactating dairy cows. Journal of Dairy Science 92: 1412-1422.

5. Souza AH, Viechnieski S, Lima FA, Silva FF, Araujo R, Bo GA, Wiltbank MC, Baruselli PS, 2009. Effects of equine chorionic gonadotropin and type of ovulatory stimulus in a time-AI protocol on reproductive responses in dairy cows. Theriogenology 72:10-21.

6. Bamber RL, Shook GE, Wiltbank MC, Santos JE, Fricke PM, 2009. Genetic parameters for anovulation and pregnancy loss in dairy cattle. Journal of Dairy Science 92:5739-5753.

7. Watters RD, Wiltbank MC, Guenther JN, Brickner AE, Rastani RR, Fricke PM, Grummer RR, 2009. Effect of dry period length on reproduction during the subsequent lactation. Journal of Dairy Science 92:3081-3090.

8. Checua CM, Beg MA, Gastal MO, Wiltbank MC, Parrish JJ, Ginther OJ, 2009. Effect of suppression of FSH with a GnRH antagonist (acyline) before and during follicle deviation in the mare. Reproduction in Domestic Animal 44:504-511.

9. Watters, RD, Wiltbank, MC, Guenther JN, Brickner AE, Rastani RR, Fricke PM, Grummer RR, 2009. Effects of dry period length on reproduction during the subsequent lactation. Journal of Dairy Science 92: 3081-3090.

10. Kneebone J, Tsang PCW, Townson DH. 2010 IDEXX SNAP® tests detect antibiotic residues in powered milk products. Journal of Dairy Science, Accepted

11. Townson DH, Putnam AN, Sullivan BT, Guo L, Irving-Rodgers HF, 2010. Expression and distribution of cytokeratin 8/19 intermediate filaments in bovine antral follicles and corpus luteum: An intrinsic mechanism of resistance to apoptosis? Histology and Histopathology 25:889-900

12. Duncan AR, 2009. Disruption of cytokeratin 18-containing intermediate filaments in bovine luteal cells: Effects on Fas expression, progesterone secretion, and FasL-induced apoptosis. MS Thesis, University of New Hampshire.

13. Forcina JJ, 2009. Genetic over-expression of cytokeratin 18 in bovine luteal cells: Effects on intermediate filament formation and Fas ligand-induced apoptosis. MS Thesis, University of New Hampshire.

14. Cushman RA, Wood JR, Slattery R, Clopton DT, Smith J, Beavers KA, Pohlmeier W, Bergman JW, Moline KV, Cupp AS (2010) Reproductive Ageing Influences Ovarian Function in Beef Cows, University of Nebraska Beef Report, p16

15. Cooney MA, Malcuit C, Banyoon C, Holland MK, Fissore RA, DCurz NT (2010) Species-Specific Differences In Activity and Nuclear Localization of Murine and Bovine Phospholipase C, Zeta 1. Biol Reprod 83(1):92-101.

16. Tripurani, S.K., Xiao, C., Salem, M. and Yao, J. 2010. Cloning and analysis of fetal ovary microRNAs in cattle. Animal Reproduction Science. 120:16-22.

17. Odhiambo JF, Poole DH, Hughes L, DeJarnette JM, Inskeep EK, Dailey RA. 2009. Pregnancy outcome in dairy and beef cattle after artificial insemination and treatment with seminal plasma or transforming growth factor beta-1. Theriogenology. 72: 566-571.

18. Inskeep K. 2009. Induction of estrus as a reproductive management tool in anestrous ewes. Proceedings Great Lakes Dairy Sheep Symposium. Ed by DL Thomas. Albany NY Nov 12-14, 2009. pp 94-110.

19. Perea-Ganchou F, Inskeep EK. 2010. Infertilidad asociada con la duracion de la fase luteal en vacas postparto. In Manejo Reproductivo de la Vaca Posparto. Ed by German E. Portillo Martinez. Cuadernos Cientificos Girarz 7:205-221. Maracaibo, Venezuala.

20. Inskeep EK. 2010. Time-dependent embryotoxicity of the endogenous luteolysin prostaglandin F2± in ruminants. Chapter 10, pp 240-255 in Endocrine Toxicology. C. Eldridge and J.T. Stevens, Eds. Informa Healthcare, London

21. Inskeep EK, Dailey RA. 2010. Maximizing Embryonic and Early Fetal Survival in Dairy Cattle. In WCDS Advances inDairy Technology 22:51-69. Lorraine Doepel, Editor.

22. Relling, AE, Pate, JL, Reynolds, CK and Loerch, SC. 2010. Effect of feed restriction and supplemental dietary fat on gut peptide and hypothalamic neuropeptide mRNA concentrations in growing wethers. J. Anim. Sci. 88: 737-748

23. Ndiaye, K, Lussier, JG, and Pate, JL. 2010. Molecular characterization and expression of DERL1 in bovine ovarian follicles and corpora lutea. Reprod. Biol. Endocrinol. 8:94 (http://www.rbej.com/content/8/1/94)

24. Pate, JL, Toyokawa, K, Walusimbi, S, and Brzezicka, E. 2010. The Interface of the Immune and Reproductive Systems in the Ovary: Lessons Learned from the Corpus Luteum of Domestic Animal Models. Amer. J. Reprod. Immunol. 64:275-286.

25. Smitz, J., R.L. Stouffer, E.E. Telfer, T.K. Woodruff, M.B. Zelinski, M.M. Dolmans, J. Donnez, J.E. Fortune, O. Hovatta, K. Jewgenow, H.M. Picton, C. Plancha, L.D. Shea, 2010. Current achievements and future research directions in ovarian tissue culture, in-vitro follicle development and transplantation: Implications for fertility preservation. Human Reproduction Update 16:395-414.

26. Fortune, J.E., M.Y. Yang, and W. Muruvi, 2010. The earliest stages of follicular development: Follicle formation and activation. Reproduction in Domestic Ruminants VII, M.C. Lucy, J.L. Pate, M.F, Smith, and T.E. Spencer (eds). Nottingham Univ. Press, Nottingham, UK, p. 203-216.