Brief Summary of Minutes of Annual Meeting

ANNUAL REPORT AND MINUTES, NE-1007 TECHNICAL COMMITTEE
June 14-15, 2004
Inn at Holiday Valley, Ellicottville, NY

The meeting was called to order by Chair David Townson (NH) @8:45 AM. The following persons were in attendance: New York: Joanne Fortune, Ron Butler, Phil Bridges, and Joe Folger, New Hampshire: Paul Tsang, Dave Townson, Ohio: Joy Pate, Massachusetts, Rafael Fissore and Chris Malcuit, West Virginia: Keith Inskeep, Administrative Advisor: Bill Vinson, USDA/CSREES: Debora Hamernik. The minutes of the previous meeting, at Storrs CN, prepared by Dr. Inskeep, as Secretary, were approved. Dr. Robert Milvae (CT) arrived late to the meeting, but a brief summary of his station report is included.

Station reports were presented by Objective.

Administrative Advisor Bill Vinson (WV) reminded the group of deadlines for the annual report and made other remarks.

A report on changes in CSREES and other national USDA news was presented by Deb Hamernik (USDA/CSREES). She brought greetings from Glenn Gray, formerly CSREES representative to this group. Several open positions were noted. Electronic submission of NRI proposals will not be implemented in 2004. Electronic review will see increased use, however. A new and earlier deadline for proposals in animal reproduction is likely for the 2005 fiscal year. Several workshops and conferences were discussed. Problems with the CSREES website relative to finding information on the NRI were discussed extensively.


Work Planned for Next Year:

OH: Complete studies of MIF in ovaries of mice and sheep.

Complete study of gene expression in luteal cells from early vs. midcycle CL, in response to cytokine treatment in vitro.

Continue studies of the role of MHC class II molecules in the CL, and begin experiments to identify potential peptides that are presented.

Examine the interaction between luteal cells and gamma delta T cells, and the potential role of gamma delta T cells in luteal function.

NH: Determine whether other heat shock proteins, e.g., HSP90, are induced in heat shocked bovine luteal cells in vitro.

Determine whether other MMPs, e.g., collagenase and stromelysin, are induced in heat shocked bovine luteal cells in vitro.

Determine the regulation of HSP expression by PGF2 alpha and TNF alpha.

Continue studies of immune cell-endothelial cell-steroidogenic cell interactions within the bovine corpus luteum and the relevance to luteolytic sensitivity to PGF

NY: Measure DNA in the granulosal cell cultures to determine whether temperature had any effect on cell numbers, to normalize the data for granulosal cells by the amount of DNA, and to prepare the results summarized above for publication.

Initiate experiments on the mechanisms of the effects of hyperthermic conditions in vitro on steroidogenesis by theca interna and granulosal cells isolated from preovulatory follicles.

Continue analyses of follicular and seasonal factors in embryonic mortality in dairy cows.

Complete work with metabolic effects of propylene glycol.

WV: Complete replication of studies of pre-insemination treatment with TGF-beta or seminal plasma in beef cows bred AI. Continue similar studies in dairy cows and in beef and dairy heifers.

Complete replication of studies of dosage of progesterone and lamb removal in out-of-season breeding of lactating ewes.

Complete replication of studies of effects of concentrations of progesterone during days 28 to 38 post-insemination on retention of pregnancy in beef cows.


A committee was appointed (David Townson and Paul Tsang) to select the site for the 2005 meeting on June 1 to 3. The meeting was adjourned at 6:10 PM.

Objective 1: Examine events surrounding follicular development and the follicular to luteal transition that may affect embryonic/fetal mortality

NY: Pregnancy (in 48 lactating cows) was associated with larger follicle diameter on the day of AI than in 95 non-pregnant, lactating cows, but the incidence of pregnancy was not different among cows with follicle diameter above or below the mean (33% vs 35%, respectively). Estradiol concentrations on day of AI were not significantly different between pregnant and non-pregnant cows in either season, nor between pregnant cows at day 63 and those losing the conceptus between d 28-63. Cows pregnant at d28 had higher progesterone concentrations from d5 post-AI compared to non-pregnant cows suggesting that a more rapid rate of post-ovulatory rise of progesterone improves embryo survival. Fifteen to 17% of cows inseminated did not meet the criteria of having progesterone less than 0.7 ng/mL on day of AI and greater than 1.0 ng/mL on day 5 post AI.

WV: Conception rates in beef cows treated (intrauterine) with seminal plasma 12 h before insemination appeared higher (78%, 25/32) than those treated with BSA (control, 61%, 17/28), and cows treated with TGF-beta 1 were intermediate (71%, 24/34). Based on these preliminary data, a total of 200 head of cattle per treatment will be required to yield an 87.5 percent chance of determining significance at p < 0.05.

Mean ovulation rates were 2.0 ± 0.2, 2.58 ± 0.2 and 2.8 ± 0.19 for ewes with high, medium and low progesterone, respectively, during the previous cycle, being significantly lower in ewes with high progesterone than in the other two groups (P<0.01).

OH: Studies of MIF in follicular development: ovaries have been collected for the project, except for lambs 240 days of age.

Objective 2: Examine mechanisms involved in insufficient or altered luteal function that may result in embryonic/fetal mortality

NY: Profiles of progesterone on days 1 (AI) through 7, 9, 14, and 22 differed
between pregnant and non-pregnant cows on the Cornell Ov-Synch protocol(described in Obj 1) in the cold season. Within the non-pregnant cows, progesterone tended to be higher in the cold than in the warm season.

OH: Subclasses of endothelial cells within the CL express class II MHC molecules, and expression varies with stage of the estrous cycle.

Luteal cell stimulation of T cell proliferation is accompanied by increased secretion of IFN-? and IL-10. Increased production of both of these cytokines precludes a conclusion of a Th1 vs aTh2 response.

Luteal cells stimulate proliferation of gamma delta T cells. These cells are not considered to be MHC restricted, which calls to question the mechanism by which these cells are activated by luteal cells. The gamma delta T cells are present in greater proportions in ruminants than in humans or rodents, thus may represent a unique regulatory cell type in these species.

Bovine lymphocytes do not express the gene for the cytosolic progesterone receptor, regardless of activation state. In contrast, bovine lymphocytes do express the membrane progesterone receptor gene, and the mRNA for this receptor is increased when the lymphocytes are activated by Con A.

WV: The mechanism by which CL in P ewes are resistant to PGF2alpha at d 12 may include down regulation of PGFS and a higher ratio of luteal PGES to PGFS. PGDH mRNA did not differ between P and NP ewes, despite greater PGDH activity in P ewes.

Bovine luteal cells are sensitive to ET-1 during early and mid luteal phase, as ET-1 elicited a change in [Ca2+]i and also inhibited progesterone biosynthesis. The sensitivity of luteal cells to PGF2 alpha assessed in terms of [Ca2+]i indicates that SLC are less responsive to PGF2 alpha than LLC, indicating a cell specific regulation by PGF2 during early CL development.

PKC beta II and epsilon were expressed differentially as a function of development of bovine CL, being greater at day 10 than at day 4. PGF2 alpha induced PKC activation in both day 4 and day 10 CL. Furthermore, PKC specific inhibitor blocked the PGF2 alpha induced rise in [Ca2+]i and antagonized the PGF2 alpha mediated decline of P4 synthesis in steroidogenic cells. Thus, differential expression of PKC isoforms may be a part of the cellular mechanism responsible for the relative insensitivity of the early CL to the luteolytic actions of PGF2 alpha, and this cellular mechanism may be mediated by the regulatory role of PKC in the PGF2 alpha induced rise in [Ca2+]i.

In beef cows with gestation supported by exogenous progesterone after removal of the corpus luteum, experimental decreases in progesterone on day 28 to 38 tended to reduce retention of pregnancy. Pregnancy appears to be sensitive to concentrations of progesterone during placentation.

NH: In co-culture, immune cells and endothelial cells can interact cooperatively to increase MCP-1 secretion in the CL, but this interaction did not affect ET-1 secretion nor was it influenced by PGF. In addition, activated immune cells impaired progesterone production by steroidogenic cells of the CL.

CT: Four doses of 50 mg ET-1 given every two hours caused complete luteolysis, strengthening the concept that ET-1 may mediate the action of PGF2?.

L-NAME, an inhibitor of nitric oxide, mitigated the luteolytic effect of ET-1, allowing only a transient decrease in progesterone. However L-Name alone had no effect on concentrations of progesterone or estrous cycle length in control ewes.

Objective 3: Determine environmental and metabolic effects on oocyte maturation, fertilization and embryonic/fetal survival

NY: Pregnancy rate in lactating dairy cows was 33% at d 28 after AI and 23% of
these were lost by d 63. Of the conceptus losses, late embryo loss by d 42 accounted for 17% (73 % of total). Follicle size at AI was similar in cows pregnant at d 63 compared to those losing the conceptus between d 28-63. Season did not affect follicle size in either pregnant cows that remained pregnant or pregnant cows that lost the conceptus. Among pregnant cows in the cold season, conceptus loss was associated with lower plasma progesterone from d 8 post-AI onward, whereas for the warm season this difference was not significant until d 30.

Drenching cows with propylene glycol daily during the transition period had a
clear beneficial effect on the postpartum metabolic profile including increases in plasma insulin and decreases in plasma NEFA and Beta-OH butyrate concentrations. These metabolic alterations failed to elicit any demonstrable effect on the reproductive parameters. There was no difference between treatments in the proportion of cows having ovulatory, atretic or cystic follicles in the first postpartum follicle wave and no difference in LH pulse frequency.

Exposure of follicular cells to hyperthermic conditions affected both basal and gonadotropin-stimulated steroidogenesis by both thecal and granulosal cells of dominant and preovulatory follicles. The inhibitory effects of elevated temperature on secretion of estradiol and androstenedione contrasted with the stimulatory effects of progesterone. Hyperthermic conditions may be inducing premature luteinization of follicular cells and affecting fertility in vivo by a similar mechanism.

NH: Heat shock treatment (42 o C) for 24 hours upregulated the expression of HSP70 without influencing MMP2 or MMP9 activity in midcycle corpora lutea (day 10-12) of Holstein dairy cows.

WV: In ewe lambs, an oral bolus of acetate or propionate caused a marked change in the A:P ratio in hepatic portal blood for at least 4 h. Plasma concentrations of progesterone after the oral bolus of either acetate or propionate began to diverge as early as 0.5 h, differed at 3 and 4 h, and were not different by 5 h, coincident with the return to baseline A:P ratios. Changes in the A:P ratio altered circulating concentrations of progesterone, presumably by reducing the metabolic clearance when the A:P ratio was decreased.

Lamb removal in combination with higher dosages of progesterone may improve estrus, ovulation, and fertility in response to ram introduction in lactating anestrous ewes.

Work Beyond Objectives

MA: Oscillations in [Ca2+]i in cow oocytes are induced by fertilization, but not by ICSI. Experiments showed that bull sperm induced [Ca2+]i oscillations in a mouse oocyte, but mouse sperm did not induce oscillations in a cow oocyte. Polyspermy, however, with either bull or boar sperm was effective, but a single boar sperm was ineffective. Conclusion is that bull sperm do have enough sperm factor to initiate oscillations during natural fertilization, but that during ICSI either bull sperm does not release adequate amounts of the factor, or that the factor is not appropriately activated. Phospholipase C? was effective and may be the sperm factor.

In mouse, oscillations in [Ca2+]i continue after fertilizing sperm is removed, so once the sperm factor is released (30 to 60 minutes), the sperm is no longer needed. Numerous lines of evidence support that phospholipase C? is the sperm factor and that it acts through IP3 and IP3R-1 and is cell cycle dependent.

NH & CT: MMP-19 is differentially expressed over the course of the estrous cycle, being highest in the early, day 4 bovine CL. Furthermore, administration of PGF2? in vivo but not in vitro increased MMP-19 mRNA expression. Instead, treatment with TNF? in vitro stimulated MMP-19 expression in luteal cells. Collectively, MMP-19 may play a critical role when the CL is undergoing intense angiogenesis and remodeling, i.e., during early development and luteal regression.

WV: Treatment of ewes with growth hormone at breeding resulted in modified placental development, which may then allow for larger weights at birth and during postnatal growth of single lambs

Progress has been made in identification of a second oocyte-specific gene in cattle.

NH: Cytokeratin 18 appears to be an important determinant of the incidence of
apoptosis in luteal cells.

Pate, J.L. 2003. Lives in the Balance: Responsiveness of the Corpus Luteum to Uterine/Embryonic Signals. Reproduction Suppl. 61:207-217.

Knights, M., Baptiste, Q.S., Dixon, A.B., Pate, J.L., Marsh, D.J., Inskeep, E.K. and Lewis, P.E. 2003. Effect of dosage of follicle stimulating hormone (FSH), vehicle and time of treatment on ovulation rate and prolificacy in ewes during the anestrous season. Small Rum. Res. 50:1-9

Cannon, M.J., Petroff, M.G. and Pate, J.L. 2003. Effects of prostaglandin F2 alpha and progesterone on the ability of luteal cells to stimulate T lymphocyte proliferation. Biol. Reprod. 69:695-700.

Cannon, M.J. and Pate, J.L. 2003. Role of Major Histocompatibility Molecules in Luteal Function. Reprod. Biol. Endocrinol. http://www.rbej.com/content/1/1/93

Davis, Tracy. 2004. Ph.D. Dissertation. Role of the Endocrine and Immune Systems in the Developing and Regressing Corpus Luteum

Townson DH, Liptak AR 2003 Chemokines in the Corpus Luteum: Implications of Leukocyte Chemotaxis. Reproductive Biology and Endocrinology 1:94.

Zhang B, Moses MA, Tsang PCW. 2003. Temporal and spatial expression of tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and -2) in the bovine corpus luteum. Reproductive Biology and Endocrinology 1:85 (7 November).

Allen JF 2003 Expression of cyclooxygenases-1 and -2, steroidogenic acute regulatory protein, and heat shock protein-70 during prostaglandin F2 alpha-induced luteolysis in sheep. MS Thesis, University of New Hampshire.

Knights, M., T. Hoehn, D. Marsh, P. Lewis, J. Pate, A. Dixon and K. Inskeep. 2003. Reproductive management in the ewe flock by induction or synchronization of estrus. West Virginia Agricultural and Forestry Experiment Station Bulletin 727.

Sen, A., J. Browning, E. K. Inskeep, P. Lewis, and J. A. Flores.
2004. Expression and activation of protein kinase C isozymes by prostaglandin F2a in the early- and mid-luteal phase bovine corpus luteum. Biol Reprod 70:379-384.

Starbuck, M. J., R. A. Dailey and E. K. Inskeep. 2004. Factors affecting retention of early pregnancy in cattle. Anim. Reprod. Sci. In Press.

Inskeep, E. K. 2004. Preovulatory, postovulatory, and post- maternal recognition effects of concentrations of progesterone on embryonic survival in the cow. J. Anim. Sci. 82(E. Suppl.):E24?E39.

Choudhary, E., B. A. Costine, M. E. Wilson, E. K. Inskeep and J. A. Flores. 2004. Prostaglandin F2 alpha (PGF2 alpha)-independent and -dependent regulation of the bovine luteal endothelin system. Dom. Anim. Endo. 27:63-79.

Bridges, P.J. and J.E. Fortune, 2003. The characteristics of developing prolonged dominant follicles in cattle. Domestic Animal Endocrinology 25:199-214.

Folger, J.K., P.J. Bridges, and J.E. Fortune, 2004. Heat stress in vitro affects steroid production by theca and granulosa cells from bovine preovulatory follicles. Reproduction, Abstract series No. 31, p. 10.

Rivera, G.M. and J.E. Fortune, 2003. Proteolysis of IGF binding proteins-4 and -5 in bovine follicular fluid: Implications for ovarian follicular selection and dominance. Endocrinology 144:2977-2987.

Fortune, J.E., 2003. The early stages of follicular development: activation of primordial follicles and growth of preantral follicles. Animal Reproduction Science 78:135-163.

Madan, P., P.J. Bridges, C.M. Komar, A.G. Beristain, R. Rajamahendran, J.E. Fortune, and C.D. MacCalman, 2003. Expression of messenger ribonucleic acid for ADAMTS subtypes changes in the periovulatory follicle after the gonadotropin surge and during luteal development and regression in cattle. Biology of Reproduction 69:1506-1514.

Fortune, J.E., G.M. Rivera, and M.Y. Yang, 2004. Follicular development: the role of the follicular microenvironment in selection of the dominant follicle. Animal Reproduction Science (in press).

Butler, S.T., A.L. Marr, S.H. Pelton, R.P. Radcliff, M.C. Lucy, and W.R. Butler. Insulin restores hepatic growth hormone (GH) responsiveness during lactation-induced negative energy balance in dairy cattle: effects on expression of insulin-like growth factor-I and GH receptor 1A. J. Endocrinol. 176:205-217. 2003.

Butler, S.T. and W.R. Butler. The effect of daily drenching with propylene glycol during the transition period on LH pulsatility and the fate of the first follicle wave in dairy cows. J. Dairy Sci. 86(Suppl. 1):239. 2003.

Butler, S.T. and W.R. Butler. Ovarian activity during the early postpartum period: The role of circulating levels of insulin, IGF-I and urea. Proc. 2003 Cornell Nutrition Conference. pp. 219-231

Butler, W.R. Energy balance relationships with follicular development, ovulation and fertility in postpartum dairy cows. Livest. Prod. Sci.. 83:211-218, 2003.

Butler, W.R. Negative energy balance may delay subsequent ovulation. Feedstuffs 75(#50):10-13, 2003.

Bacon, C., C.S. Keator, R.A. Milvae and J. A. McCracken. 2004. Effect of depleting luteal oxytocin on pregnancy rates in sheep. Biol. Reprod. 70:Suppl. 1:344.

Keator, C.S., D.T. Schreibner, J. A. McCracken and R.A. Milvae. 2004. In vivo effect of a nitric oxide inhibitor (L-NAME) on PGF2a-induced luteolysis in sheep. Biol. Reprod. 70:Suppl. 1:455.

Zhang, B., H. Zhang, R.A. Milvae, C. Keator, M.A. Moses and P.C.W. Tsang. 2004. Tissue inhibitor of metalloproteinases 3 (TIMP-3) and its regulation by tumor necrosis factor in the bovine corpus luteum. Biol. Reprod. 70:Suppl. 1:abst