Source: OREGON STATE UNIVERSITY submitted to NRP
EVALUATION OF A SINGLE LUTALYSE INJECTION PROTOCOL TO REDUCE UTERINE INFECTIONS AND IMPROVE REPRODUCTIVE EFFICIENCY IN POSTPARTUM DAIRY COWS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
ANIMAL HEALTH
Reporting Frequency
Annual
Accession No.
0229915
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
May 15, 2012
Project End Date
Sep 30, 2013
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331
Performing Department
Animal and Rangeland Sciences
Non Technical Summary
Uterine infections during the period after calving, or the postpartum period, are a significant problem to dairy producers. Dairy cows are particularly susceptible to develop uterine infections because of conditions associated with housing and production. Bacterial infections, such as endometritis, metritis and pyometra, occurring within 28 days postpartum affect 10 to 50% of cows annually. Fertility is reduced in cows with uterine infections because of abnormal reproductive cycles, lower conception rates (20% lower than healthy cows), and longer days open (an average of 30 days longer than healthy cows). The cost of days open beyond 100 days has been estimated to be as much as $5.40/day for the dairy cow. Depending on the uterine infection incidence one uses to calculate cost to Oregon dairy producers on a state-wide basis, income loss is estimated to range between $2 million to 10.1 million annually. During pregnancy, the uterus is a sterile environment, but during calving and the early postpartum period, bacteria from the cow's environment can colonize the uterus, causing infection. It is known that uterine susceptibility to bacterial infection is higher when the ovarian hormone progesterone is high and is lower when the ovarian hormone estrogen and the uterine hormone prostaglandin F2a (PGF) are high. These findings, though important, have not aided in the development of new methods of prevention and treatment of uterine infections. Significant research effort has been directed towards studying the pathogenic microorganisms that cause uterine infections. However, despite this research, preventative practices and new treatment strategies have not been developed, and the incidence of uterine infection has not changed significantly in cattle over the previous 30 years. New knowledge in this area is a necessary first step towards development of new and practical methods for preventing uterine infections and novel therapeutic regimes not requiring antibiotics for treatment. Our laboratory demonstrated that a single injection of Lutalyse (the pharmaceutical product of PGF) increased the number of circulating neutrophils within one hour after injection and for several hours thereafter. In a second experiment, Lutalyse injections on either Days 0-1 and 14 or Days 14 and 28 postpartum increased neutrophils and decreased total bacteria in the uterus and decreased days open and services per conception. The greatest effect was observed in cows injected on Days 0-1 and 14. Endogenous PGF would be high around parturition (Day 0) but low by Day 14. The Day 14 Lutalyse injection may function to maintain high plasma PGF in the early postpartum period most susceptible to uterine infections. Therefore, our hypothesis is a single injection of Lutalyse on Day 14 postpartum will increase uterine neutrophils and decrease uterine bacterial load thereby decreasing days open and services per conception. Expected outcomes and impacts include a change in knowledge, where the results of this basic research will be published in a scientific journal, and a potential change in conditions in better and less expensive animal health.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113410102050%
3113410109020%
3113410110020%
3113410116010%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1090 - Immunology; 1160 - Pathology; 1100 - Bacteriology; 1020 - Physiology;
Goals / Objectives
Maintenance of uterine health during the period after calving, or the postpartum period, is a significant challenge for dairy producers. Fertility is poorer in cows with uterine infections and in Oregon this reduced fertility can contribute to an income loss estimated to reach as high as $10 million annually. Uterine resistance to bacterial infection seems to be greater when the uterine hormone prostaglandin F2a (PGF) is high. Our laboratory demonstrated that a single injection of Lutalyse (the pharmaceutical product of PGF) increased the number of circulating neutrophils within one hour after injection and for several hours thereafter. In a second experiment, Lutalyse injections on either Days 0-1 and 14 or Days 14 and 28 postpartum increased neutrophils and decreased total bacteria in the uterus and decreased days open and services per conception. The greatest effect was observed in cows injected on Days 0-1 and 14. Endogenous PGF would be high around parturition (Day 0) but low by Day 14. The Day 14 Lutalyse injection may function to maintain high plasma PGF in the early postpartum period most susceptible to uterine infections. A single Lutalyse injection on Day 14 may be the simplest and most efficacious preemptive strategy for dairymen to counter uterine infections and improve uterine health and fertility. Therefore, the objective of this proposal is to evaluate the effects of a single Lutalyse injection administered on Day 14 postpartum on uterine bacterial and neutrophil populations and numbers of days open and services per conception in postpartum dairy cows. Expected outputs: Activities - conducting and analyzing experiments in the research project and teaching and mentoring graduate students; Events - presenting the results of the research at scientific societal conferences and training graduate students in this research; Products - graduating students with master's or doctoral degrees.
Project Methods
Eighty postpartum cows at the OSU Dairy Center will be randomly assigned to two treatments (forty cows per treatment) and injected with either saline (5 ml; im) or Lutalyse (25 mg/5 ml; im) on Day 14 postpartum (where Day 0 = day of calving). On the day of the injection, the cow's uterus will be sampled for uterine bacterial load using a double guarded swab (Time 0). Twenty-four h after the first swab, a second uterine swab will be collected (Time 24). To provide an assessment of the uterine neutrophil population, a guarded CytoBrush will be passed into the uterus immediately following the uterine swabbing at Times 0 and 24. The CytoBrush will be rolled onto a microscope slide to create a cell smear and neutrophils will be counted. Blood samples (10 ml) will be drawn by coccygeal venipuncture at Times 0 and 24 to assay plasma progesterone concentrations. Serial dilutions will be made from the original uterine sample and 100 ul will be plated onto each side of a Blood Agar/MacConkey's Agar bacteriological culture biplate. Colony counts on the blood agar side provide the total number of pathogenic bacteria and counts from the MacConkey's agar side provide the number of E.coli. All cows on experiment will be evaluated daily for evidence of uterine infections, e.g., vaginal discharge, fever, etc. Herd records will be evaluated in a timely fashion and data pertaining to days open and services per conception will be collected for analysis. Differences due to treatment in the total numbers of bacteria, E. coli and neutrophils and plasma progesterone will be analyzed by repeated measures analysis of variance (ANOVA). If significant effects are observed in the ANOVA, differences between means will be evaluated by Fisher's least significant differences procedures. Observational data, e.g., number of cows observed with uterine infections, will be analyzed for differences using Chi-square procedures. Differences due to treatment in the numbers of days open and services per conception will be evaluated using the pooled t-test. Lutalyse treatment should increase uterine neutrophils and decrease uterine bacterial load thereby improving uterine health and decreasing days open and services per conception. Significant savings can be realized by dairy producers implementing such a protocol because of the reduced expense associated with fewer days open and reduced labor and semen costs associated with fewer services per conception. A functional pre-emptive strategy would also decrease antibiotic use, further reducing drug costs. Efforts will include adding this information into material used for formal classroom and laboratory instruction and outreach. Lutalyse is commonly used in dairy operations to synchronize estrous cycles for artificial insemination and a single dose is under $3.00. Therefore, this protocol can be easily implemented with relatively little cost. It is anticipated the results of this research will be published in a format accessible to dairy producers for information. The success and merit of the study will be evaluated by tracking the number of times the publication reporting this research is cited in the scientific literature.

Progress 05/15/12 to 09/30/13

Outputs
Target Audience: Target audiences include: dairy cattle producers and veterinarians who would apply these techniques to their herds, educators who would learn of these results and disseminate the information to their students, and students in animal and veterinary sciences to whom this information would be delivered. Efforts Efforts include adding the research results to formal classroom and laboratory instruction and workshops for extension presentations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training activities: The graduate student involved in the projectparticipated in the project design and analysis and interpretation of the results. The graduate student was trained in: drawing and assaying blood samples, uterine swabbing, counting neutrophils, culturing and counting bacteria and recording and managing data. Undergraduate students who worked on the project were trained in blood collections and assisted the graduate student in her bench work. Professional development: The graduate student presented the results of the research at a departmental seminar. How have the results been disseminated to communities of interest? Results of the research were published in the OSU Beef Research Report which is accessible to cattle producers inhard copy format orthrough the Oregon State University-Beef Cattle Sciences website. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? IMPACT: Uterine infections during the period after calving are a costly problem for dairy producers. Fertility is poorer in cows with uterine infections and in Oregon this reduced fertility can contribute to an income loss estimated to reach as high as $10 million annually. White blood cells, especially neutrophils, are the first line of defense against uterine infections. Preliminary experiments conducted in our laboratory investigated the effect of a single injection of the pharmaceutical product, Lutalyse, a drug commonly used in dairies for reproductive purposes, on plasma neutrophil numbers in dairy cows. Lutalyse caused the number of circulating neutrophils to increase within one hour after administration and remain elevated for four hours thereafter. These data suggest that Lutalyse recruits neutrophils into the cow’s plasma. This property of Lutalyse could be used to create a preemptive strategy that counters the development of uterine infections in dairy cattle. Therefore, this research evaluated the effects of a single injection of Lutalyseadministered14 days postpartum on uterine neutrophil and bacterial populations and numbers of days open and services per conception indairy cows. Cows in this study were from a dairy containing 3000 Holstein cows with 1500 in the milking string. Cows were supplied with water using a free-choice system. The ration fed included corn silage, grass silage, alfalfa, corn, cotton seed and soy bean meal. Postpartum cows were given a 55-day voluntary waiting period before synchronization and artificial insemination. Pregnancy was detected by a veterinarian through rectal palpation and ultrasound at 40 days of gestation. Ovarian structures were also palpated in the event that pregnancy did not occur in order to identify whether inability to conceive was the result of failure of resumption of the estrous cycle. A total of forty cows at 14 days postpartum were given an intramuscular injection of either PGF2α (Lutalyse) or saline. The first cow was chosen at random for injection of either Lutalyse or saline and subsequent cows were injected with alternating treatments to ensure randomization. Uterine bacteria samples were obtained at 0 and 24 h after injection through rectal palpation and swabbing the inside of the uterine body. For the 0-h sample recovery, the swab was conducted just before the injection was administered. To recover bacteria, the uterus was first palpated rectally, and once the cervix was grasped securely, the vulva was cleaned using paper towels and spread open and the first guarded tube of the swab was inserted into the vagina. This tube was inserted into the external cervical os and the second guarded tube was pushed thru the first tube into and through the cervix. Once the second guarded tube was in the uterine body approximately 2 cm, the swab was then pushed through the second guard tube and spun gently on the uterine body. The swab was then retracted back into the second guarded tube and both guarded tubes were removed from the vagina. The swab with the uterine sample was broken off and placed in a tube containing ten ml of Dulbecco’s phosphate buffered saline (DPBS). Samples were brought to the lab and a 1/10 dilution was made from the sample collected in the field using DPBS as the diluting fluid. One hundred microliters of each dilution (0 and 1/10) was spread onto each side of a Blood/MacConkey Agar bacteriological culture biplate. The biplates were covered and left facing up for one h at room temperature and placed face down in an incubator at 37°C for 24 h. The numbers of colonies that grew on either side of the biplates were counted and recorded. Services per conception and days open were recovered from breeding records maintained for the cows in the experiment. Differences in numbers of bacteria between the 0 and 24-h swabbing, services per conception and days open were analyzed using one-way ANOVA with treatment (Lutalyse or saline) as the main effect. Changes in uterine bacterial populations did not significantly differ between cows injected with Lutalyse or saline. However Lutalyse treatment reduced total bacteria by 30 cells compared to 3 cells in cows injected with saline.The dairy at which this study was performed chose to rebreed cows with exceptional milk production as many times as was financially feasible. It is for this reason some data for cows included beyond 2 or 3 services per conception. However this gave a clearer picture of reproductive performance for each cow. There was no significant difference in services per conception between cows injected with Lutalyse or saline however cows injected with Lutalyse had 0.2 fewer services per conception. Number of days open for these cows was also not significantly differentbetween cows injected with Lutalyse or saline. However, cows injected with Lutalyse had 15 fewer days open compared to cows injected with saline. Dairy producers implementing this protocol would experience reduced costs associated with breeding and using Lutalyse would reduce the overall need for antibiotics. Change in knowledge - Results of this basic research will be published in a scientific journal Change in action – Adoption and use of new methods Change in conditions - Better and less expensive animal health; higher productivity in food provision

Publications

  • Type: Other Status: Published Year Published: 2014 Citation: Younger, K.G., Lulay, A.M., and Menino, Jr., A.R. 2014. Effects of prostaglandin F2a on reproductive performance in dairy cows. Oregon State University-Beef Research Report