SHORT PROGESTERONE PRIMING PROTOCOL FOR ESTRUS SYNCHRONIZATION IN THE GOAT DURING THE NON-BREEDING SEASON

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0231437
• Project No.: MASV-FFGOATS
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2017

Project Director
Ayres, SA.

Recipient Organization
TUFTS UNIVERSITY
200 WESTBORO ROAD
N. GRAFTON,MA 01536

Performing Department
Biomedical Sciences

Non Technical Summary
As goats are seasonal breeders, protocols have been developed to bring animals into heat (estrous synchronization) during the non-breeding season, in order to produce goat meat and milk year round. All estrous synchronization protocols for breeding out of season require administering the hormone progesterone, commonly via a vaginal insert known as a Controlled Internal Drug Releaser (CIDR). New protocols that include insertion of the CIDR for as little as 3-5 days (short progesterone priming protocols) can effectively bring animals into heat to be bred, but the pregnancy rate is considerably lower than when the same protocol is used during the breeding season. Work in our laboratory has suggested that the levels of serum progesterone from CIDRs during the non-breeding season are relatively low, and that the animals with the highest levels of serum progesterone were the most likely to become pregnant. We therefore hypothesize that increasing the serum progesterone levels in goats undergoing short progesterone priming for estrous synchronization during the non-breeding season will result in an increase in pregnancy rate. To test this hypothesis we propose two experiments. The first experiment will compare the use of modified cow CIDRs with small ruminant CIDRs to increase the level of serum progesterone during the non-breeding season. Previous work in our laboratory has shown that 1/3 of a cow CIDR inserted into the goat vagina produced serum progesterone levels similar to those produced by small ruminant CIDRs (3-5 ng/ml). We hypothesize that inserting 2/3 of a cow CIDR into the goat vagina should produce serum progesterone levels of approximately 6-10 ng/ml. Twelve animals will be divided into 3 groups of 4 animals each, and will receive either 1/3 of a cow CIDR, 2/3 of a cow CIDR, or a small ruminant CIDR for 3 days. Blood will be collected for serum progesterone analysis before, during, and after CIDR insertion. The experiment will be run two more times such that each animal will be exposed to all three CIDR types. The second experiment will compare the progesterone levels and pregnancy rates of goats that have undergone estrous synchronization protocols using CIDRs with high levels of progesterone (n=5) versus CIDRs with low levels of progesterone (n=5) and controls without CIDRs (n=2). Briefly the protocol involves insertion of the CIDR for 3 days, injection of FSH on Days 2 and 3 (induce follicles to develop) and injection of GnRH (induce ovulation) 2 days after CIDR removal, then breed all animals. Blood samples will be taken during estrous synchronization and for 4 weeks after breeding. High levels of serum progesterone for more than 25 days will be considered indicative of pregnancy. This experiment will be run twice, with the high and low groups switched. We anticipate that modified bovine CIDRs can be used to increase serum progesterone levels during a short progesterone priming protocol for estrous synchronization, and will result in increased rates of pregnancies. If pregnancy rates can be improved, this protocol could be effectively used by producers to breed goats outside of the breeding season.

Animal Health Component

100%

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
301	3820	1050	100%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3820 - Goats, meat, and mohair;

Field Of Science
1050 - Developmental biology;

Keywords

goats

estrous synchronization

progesterone

cidr

short protocol progesterone priming

anestrous breeding

out of season breeding

Goals / Objectives
Estrous synchronization during the non-breeding season (anestrus) in goats is an important component of herd management in order to produce meat and milk year round. An essential part of estrous synchronization is progesterone priming. Older synchronization protocols use long periods (9-21 days) of exogenous progesterone administration in an attempt to "mimic" or replace the production of progesterone by the corpus luteum (CL). With increased knowledge of the dynamics of follicular waves and the role of the hypothalamic-pituitary-gonadal axis, it has been possible to design protocols with shorter periods of exogenous progesterone priming (5-7 days). By decreasing the time of exogenous progesterone administration, herds can be synchronized more quickly, resulting in reduced costs for producers. Although 5-day short progesterone priming protocols have been used successfully to induce and synchronize estrus, the resulting pregnancy rates during the non-breeding season have been lower than those during the breeding season. The cost of using estrous synchronization protocols out of season cannot be justified if the resulting pregnancy rates are too low. There is therefore a need to develop short P4 priming protocols that produce better pregnancy rates during the non-breeding season. Our laboratory is well suited to address this need, as we have been investigating the effect of assisted reproductive techniques (ARTs) on the quality of oocytes and embryos in ruminants. Our long term goal is to investigate and understand the effects of estrous synchronization and superovulation protocols on oocytes and embryos, so that these protocols can be adjusted to maximize fertility while minimizing administration of exogenous substances to the animals. The objective of this project is to take a three day progesterone priming protocol developed in our laboratory and test the effects of different levels of serum progesterone, produced by exogenous progesterone administration, upon subsequent fertility in the goat during the non-breeding season. Work in our laboratory has led us to hypothesize that during the non-breeding season, higher blood levels of progesterone produced during priming are related to subsequent increases in fertility. This could be the result of some effect directly or indirectly on the developing oocyte, or an effect on the follicular cells, particularly those that will go on to form the corpus luteum. We plan to test our hypothesis with the following two specific aims. The first is to establish a method to increase serum progesterone levels in the goat using a modified cow CIDR. Previous work in our laboratory has shown using 1/3 of a bovine CIDR (Eazi-Breed CIDR Bovine; Pharmacia and Upjohn, New York, NY) will produce serum progesterone levels of 3-5 ng/ml. By using 2/3 of a bovine CIDR we estimate that serum progesterone levels could be increased to approximately 6-10 ng/ml. The second specific aim is to compare the pregnancy rates of goats receiving high versus low levels of exogenous progesterone in a short priming protocol during the non-breeding season.

Project Methods
Study 1: Testing serum progesterone levels produced by modified bovine CIDRs compared to small ruminant CIDRS. During the early non-breeding season, 12 goats will be divided into three groups. Group A (n=4) will receive a small modified bovine CIDR (1/3 of bovine CIDR), Group B (n=4) will receive a large modified bovine CIDR (2/3 of a bovine CIDR) and Group C (n=4) will receive a small ruminant CIDR. CIDRs will remain in place for three days. Blood will be taken for 2 days prior to insertion, 3 days during insertion and for 4 days after CIDR removal. Serum will be collected and frozen at -80 degrees Celsius for later evaluation. After a 5 day "washout" period the study will be repeated with each group receiving a different type of CIDR. After a final 5 day "washout period the study will be repeated again such that each group is exposed to all three CIDR types. Serum progesterone levels will be determined using a progesterone radioimmunoassay kit (Coat-A-Count Progesterone RIA, Diagnostic Products Corporation, Los Angeles, CA, USA). The progesterone levels from the three groups will be compared. Study 2: Comparing pregnancy rates of goats during the non-breeding season using short progesterone priming protocols that produce high or low serum progesterone levels. Later in the non-breeding season, 12 goats will be divided into three groups. Group A (n=5) will receive the short progesterone priming protocol using a large modified bovine CIDR. Group B (n=5) will receive the short progesterone priming protocol using a small ruminant CIDR. Group C (control, n=2) will receive the short progesterone priming protocol without any exogenous source of progesterone. Briefly, the protocol is as follows. Either small ruminant or bovine CIDRs will be inserted into the vagina for three days. Follicle stimulating hormone (FSH, Follitropin-V; Bioniche, Belleville, Ontario, Canada) will be administered on the second and third days of progesterone priming (32 mg IM, SID). Prostaglandin F2alpha (Lutalyse; Pfizer, New York, NY) will be given (5 mg IM) on the day of CIDR removal to eliminate any corpora lutea (CLs). GnRH (Cystorellin; Merial, Duluth, GA) will be given (50 mcg IM) two days post CIDR removal to induce ovulation. All does will be naturally bred by the same buck at the first sign of estrus and every twelve hours thereafter until they are no longer receptive. Blood will be drawn daily during the priming protocol and twice weekly during the first 4 weeks after breeding to determine serum progesterone (P4) levels. At this point all animals will receive 5 mg of prostaglandin F2α and after a one week "washout" period the study will be repeated with Group A receiving a small ruminant CIDR and group B receiving a small ruminant CIDR. The rates of estrus induction breeding, and progesterone levels will be compared. Sustained high progesterone levels for > 25 days will be used as a criterion for pregnancy. Pregnancy rates among the groups will be compared relative to serum progesterone levels.

Progress 10/01/12 to 09/30/17

Outputs
Target Audience:Owners/veterinarians interested in non-surgical methods of sterilization Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project was carried out by veterinary students as part of a summer research project under the direction of Dr. Ayres. How have the results been disseminated to communities of interest?The preliminary data was carried out by a veterinary student as part of the school's student research day seminars. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have shown that in goats, providing exogenous progesterone in the form of small ruminant controlled internal drug releasing devices (CIDR)(EAZI-BREED™ CIDR G®, Zoetis Services LLC ) during the non-breeding season, can lead to an increase in goats exhibiting estrus. There was no difference between the 3-day and the 5-day CIDR protocol in the number of females exhibiting estrus. Therefore, it appears that protocols using short periods of exogenous progesterone exposure can be used effectively in goats during the non-breeding season.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Abstract: Ayres, SL, Chevett KL, Porter C, Blash, S, Gavin, W. Comparison of estrus, breeding and pregnancy rates in goats during the non-breeding season using a short P4 protocol with and without GnRH or hCG. Clin.Theriogenology, 7(3):332, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Invited Research Lecture: Comparison of Estrus, Breeding and Pregnancy Rates in Goats During the Non-breeding Season Using a Short P4 Protocol With and Without GnRH or hCG, Annual Meeting of the Society for Theriogenology, San Antonio, TX, August 7, 2015.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Owners/veterinarians interested in non-surgical methods of sterilization Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project was carried out by a veterinary student as part of a summer research projcet under the direction of Dr. Ayres How have the results been disseminated to communities of interest?This preliminary data was presented as part of the school's student research day seminars. What do you plan to do during the next reporting period to accomplish the goals?We are hoping to support another student project in this area of research.

Impacts
What was accomplished under these goals? The past year's activity under this project was intended as a preliminary study. Ovary slices were evaluated for follicle staining and number, approximately four to five slices were evaluated for each ovary. Two ovaries from separate animals were evaluated for each cycle stage'. While this small sample cannot provide statistically significant data, it provides a starting point for further evaluations of this data. This research remains incomplete but there are tissues available to be processed with the lessons learned in this attempt. With greater care and a larger number of samples evaluated it is possible to pull statistically significant and tangible findings out of this line of research.

Publications

Progress 10/01/14 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems:The goat herd at Tufts had been reduced, so that future work will have to be conducted using herds outside of the Veterinary School campus. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The reports for this project were completed. The student involved in the project is finishing her veterinary degree, and will be taking some time in Spring of 2016 to construct a website on Caprine Mucopolysaccharidoisis IIID and work on a publication.

Publications

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The target audience for this project is goat producers, and public/academicians interested in small ruminant reproduction, particularly out of season breeding. Changes/Problems: Caprine Mucopolysaccharidosis IIID is being investigated. What opportunities for training and professional development has the project provided? a. This project enabled a veterinary student to be involved in designing a project, collecting and analyzing data, and writing up a report in the style of a journal article. The student's work was presented at the Annual Conference for the Society for Theriogenology, and an Abstract was published in Clinical Theriogenology. This project enabled me to continue my research in the area of out of season breeding in domestic goats. b. This project enabled a veterinary student to design a research project, collect and analyze data, write up the report in the style of a journal, and present the data at Tufts Student Research Day, and at the American Dairy Goat Association National Convention. How have the results been disseminated to communities of interest? a. The current work was presented at the Annual Meeting of the Society for Theriogenology in August 2014, and was published as an Abstract in Clinical Theriogenology. b. The current work was presented at the American Dairy Goat Association National Convention in October 2014. What do you plan to do during the next reporting period to accomplish the goals? a. Hormonal assays are being performed to determine serum progesterone levels to determine rates of ovulation, and levels of progesterone during early pregnancy. b. Some goats with the homozygous condition may be followed to determine the symptoms, course and severity of the disease

Impacts
What was accomplished under these goals? 1. Major Activities Completed: a. A research project comparing two hormones to induce ovulation in goats during the non-breeding season as part of a short progesterone protocol was conducted in a larger group of animals. b. Research was started to investigate Caprine Mucopolysaccharidosis IIID in Nubian Goats 2. Specific Objectives Met: a. The Short P4 study enabled us to compare the pregnancy rates in 3 groups of goats using either GnRH, hCG or no hormones to induce ovulation. Our hypothesis was since the hypothalamic-pituitary-gonadal axis is not active during the non-breeding season, a hormone that induces ovulation at the local (ovarian) level will produce greater ovulations (and subsequent pregnancies) than a hormone that acts at the CNS (hypothalamic) level. b. A survey of New England Nubian goats was performed and blood samples were taken to test for the MPSIIID mutant allele gene. Milking records from tested animals were compared. 3. Significant Results a. The hypothesis was not supported. Group % In Estrus/bred % Pregnant US (35 days) % Delivering Number of kids % Overall Preg Rate GnRH (n=9)* 66 (6/9) 50 (3/6) 33 (1/3) 4 11(1/9) hCG (n=10) 90 (9/10) 33 (3/10) 33(1/3) 2 10 (1/10) Control (n=9)* 55 (5/9) 60 (3/5) 40(2/5) 4 22(2/9) * 1 animal removed due to illness There were no statistical differences among the groups for any parameter. Work is ongoing to look at blood serum progesterone levels to compare rates of ovulation, and progesterone levels during early pregnancy b. Blood samples were tested for 388 Nubian goats in New England. The prevalence of the MPSIIID gene was14.5% heterozygous and 0.5% homozygous recessive. Presence of the gene was not correlated to milk production. 4. Key Outcomes a. The major accomplishment of this project was to demonstrate that pregnancy rates of goats (cycled outside of the breeding season using a short progesterone protocol) were not affected by hormones normally used to induce ovulation. b. The Nubian goat survey identified a small number of Nubian goats with the mutant allele in New England, and demonstrated that this gene was not related to increased milk production.

Publications

• Type: Other Status: Published Year Published: 2014 Citation: ABSTRACT: Ayres, S.L. and Chevett, K.L. Fertility in goats during the non-breeding season using a short-term progesterone priming protocol with either GnRH or hCG to induce ovulation. Clin. Theriogenology 6(3):386, 2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: INVITED RESEARCH LECTURE presented by Sandra Ayres Fertility in Goats During the Non-breeding Season Using a Short-term Progesterone Priming Protocol with Either GnRH or hCG to Induce Ovulation, Annual Meeting of the Society for Theriogenology, Portland, OR, August 7, 2014.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: The target audience for this project is goat producers, and public/academicians interested in small ruminant reproduction, particularly out of season breeding. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project enabled a veterinary student to be involved in designing a project, collecting and analyzing data, and writing up a report in the style of a journal article. In addition, this student presented a Poster at the Merial Veterinary Scholars Symposium as well as presenting her previous summer’s work at the Annual Conference for the Society for Theriogenology. This project enabled me to continue my research in the area of out of season breeding in domestic goats. How have the results been disseminated to communities of interest? The current work was presented at the Merial Veterinary Scholars Symposium in August 2013 and is being submitted to the Society for Theriogenology as a Competitive Scientific Abstract. What do you plan to do during the next reporting period to accomplish the goals? A project has been planned to repeat the work using hCG to induce ovulation, while collecting serum and performing ultrasounds three times per week post-breeding for 6 weeks to monitor early CL and fetal development.

Impacts
What was accomplished under these goals? 1. Major Activities Completed: A research project comparing two hormones to induce ovulation in goats during the non-breeding season as part of a short progesterone protocol was conducted. 2. Specific Objectives Met: This study enabled us to compare the pregnancy rates in 2 groups of goats using either GnRH or hCG to induce ovulation. Our hypothesis was since the hypothalamic-pituitary-gonadal axis is not active during the non-breeding season, a hormone that induces ovulation at the local (ovarian) level will produce greater ovulations (and subsequent pregnancies) than a hormone that acts at the CNS (hypothalamic) level. 3. Significant Results Group GnRH (n=6): % In Estrus: 100 (6), % Bred: 33.3 (2), % Bred and Ovulated#: 100 (2), % Bred and Pregnant*: 50 (1) Group hCG (n=6): % In Estrus: 100 (6), % Bred: 100 (6), % Bred and Ovulated#: 83.3 (5), % Bred and Pregnant*: 80 (4) Control Group (n=2): % In Estrus: 0 (0), % Bred: 0 (0), % Bred and Ovulated#: 0 (0), % Bred and Pregnant*: 0 (0) #Ovulation was determined by measuring serum progesterone *Percent pregnant out of those animals that ovulated Calculated in another way, the percentage of pregnant animals in each group was 16.7% (1/6) for animals induced to ovulate with GnRH, 66.7% (4/6) for animals induced to ovulate with hCG, and 0% for control animals that were not cycled. 4. Key Outcomes The major accomplishment of this project was to demonstrate that pregnancy rates of goats (cycled outside of the breeding season using a short progesterone protocol) could be increased by using a hormone (hCG) that acts directly on the ovary to induce ovulation rather than a hormone (GnRH) that acts via the CNS.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Lecture for University of Massachusetts Animal Science Students: Assisted Reproductive Technologies in the Goat, Animal Science Department, UMass, Amherst, October 9, 2012.
• Type: Other Status: Published Year Published: 2013 Citation: Data from this project was published as an abstract: Chevett, K.L and Ayres, S.L. Efficacy of short progesterone protocol on previously anestrous does. Clin. Theriogenology 5(3):390, 2013.