EVALUATION OF ARTIFICIAL INSEMINATION IN SHEEP WITH LIQUID SEMEN

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013424
• Project Start Date: Oct 1, 2017
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF THE VIRGIN ISLANDS
(N/A)
ST. CROIX,VI 00850

Performing Department
Agricultural Experiment Station

Non Technical Summary
Thsi project will evalaute dilution of ram semen for use in transvaginal artifical insemination as a cooled liquid, as opposed to using a cryopreservation method. The protoocl could be used by local producers without a major investment in supplies, technology or requiring significant advanced training .

Animal Health Component

60%

Research Effort Categories

Basic

20%

Applied

60%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3610	1020	70%
301	3610	1081	10%
307	3610	1020	20%

Knowledge Area
307 - Animal Management Systems; 301 - Reproductive Performance of Animals;

Subject Of Investigation
3610 - Sheep, live animal;

Field Of Science
1081 - Breeding; 1020 - Physiology;

Keywords

hair sheep

semen dilution

artificial insemination

Goals / Objectives
The project will address two objectives to establish a viable liquid semen AI system for use in sheep production in the USVI:Evaluate the in vitro quality of liquid extended semen stored at 5°CEvaluate fertility of liquid-stored semen using vaginal AI

Project Methods
Objective 1:Experiments will be conducted using semen collected by artificial vagina from rams maintained at the UVI-AES Sheep Research Facility. Rams to be collected will include sexually mature (> 1 yr of age) St Croix White (n = 10) and Dorper x St Croix White (n = 10). The rams will be trained to serve an artificial vagina with the aid of estrus synchronized teaser ewes. The ewes will be synchronized using 12-d treatment with CIDR devices followed by injections with prostaglandin F2α (Lutalyse®; 15 mg, i.m.) on day of CIDR removal. Two successive ejaculates will be collected from each ram. Semen will be processed for individual rams, and the two ejaculates per individual will be pooled. Only ejaculates with a volume >0.5 mL and >70% motile sperm (subjective evaluation) will be used for processing. Concentration will be determined in PBS- glutaraldehyde diluted sperm, using a hemocytometer counting chamber. Samples will be maintained at 27°C following initial evaluation, and extended and processed at this temperature.The semen extender will consist of 5% egg yolk and 95% skim UHT milk by volume (Wildeus, 2012, 2013; Jacques et. al, 2013; Wildeus, 2014; Wildeus and O'Brien, 2016, Wildeus et. al, 2017). No antibiotics will be added to the extender because the extended semen will be cooled and only stored for 48 hr. Semen of satisfactory quality (>0.5 ml volume; >70% motile) will be extended to a final concentration of 250 million/ml in a one-step dilution with the extender, and packaged into 0.5 ml straws and stored horizontally in a Styrofoam box in a refrigerator at 5°C. In-vitro semen analysis of processed semen will be conducted immediately after packaging and at 12, 24, 48 hours after storage. Analysis will include motility and viability (Giemsa staining) immediately after warming and after incubation at 37°C for 6 hours.Extended semen samples will also be shipped to Virginia State University (VSU) for analysis. Shipments will be made using a Styrofoam box within a Styrofoam box to allow gradual cooling of samples during shipment. Cooling will be provided through the inclusion of refrigerant gel packs in the outer shipping container. An initial set of shipments will be made with extender-only straws to evaluate cooling curves and temperature holding at the sample level for boxes of varying wall thickness and for of different temperature ratings. The shipping environment at the sample level will be monitored via data loggers (Onset Computer Corp., Bourne, MA). After appropriate shipping assemblies have been identified for 5°C and 15°C environments, processed semen will be shipped for each experiment and AI period to VSU and evaluated for post shipping quality using a computer assisted sperm analyzer (CASA) system.Objective 2:St Croix White (n = 30) and Dorper x St Croix White (n = 30) ewes will be synchronized for estrus using procedures described in Objective 1, and inseminated with semen extended and stored according to the results obtained under Objective 1. Semen from a minimum of 4 rams per breed will be used. Insemination trials will be conducted in February, June and October during the established breeding periods in the accelerated lambing system used at UVI (Godfrey, 2005; Godfrey et. al, 2010). Ewes will be randomly allocated, balanced by breed, to be inseminated at either 12 or 36 hours after first incidence of standing heat or at 48 hours after CIDR removal. Semen for AI will be collected twice daily during the insemination period and stored for 12 hours before use.Inseminations will be carried out by deep vaginal deposition of semen without the use of a speculum in restrained ewes, using a standard 0.5 ml AI gun. Ewes will be inseminated with the semen from a like breed ram. Clean-up rams will be placed with ewes 10 days after the last day of insemination with rams of like breed. Pregnancy success in response to artificial insemination will be determined by transrectal ultrasonography 30 days after the last day of insemination; a second confirming ultrasound examination will be conducted 45 days after the last day of insemination. Lamb survival, litter size, and lamb birth weight will be recorded at lambing.Statistical AnalysisData will be analyzed using General Linear Model procedures (SAS 9.4). Semen quality traits will be analyzed using breed of ram and time of storage as the main effects. Fertility to AI will be analyzed using Chi Squared analysis between breeds. Lamb traits will be analyzed using breed, dam age and parity, type of birth (single vs multiple) and sex of lamb as independent variables.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience: Nothing Reported Changes/Problems:Covid-19 restricted access to the UVI campus and off campus research facilities with government stay at home orders in March. 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?Depending on COVID restrictions semen will be collected from rams and the efficacy of using an extender to store liquid semen will be evaluated.

Impacts
What was accomplished under these goals? No data was collected due to COVID restrictions.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 125. Godfrey, R.W., S. Keiper and S.A. Lakos. 2020. Evaluation of extended hair sheep ram semen stored as liquid at 5�C. Presented at the Southern Section of the American Society of Animal Science, January 25-28, 2020, Chattanooga, TN.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Sheep producers in the Caribbean and mainland U.S. Changes/Problems:The use of an artificial vagina to collect semen from the rams proved to be problematic as our rams were not trained for that procedure and collectiosn were difficult. We found an intravaginal semen collection vessel in the literature and spoke with the authors of the article about it and then developed our own version of the device for our use. It worked very well and we will continue to use it in future semen collection trials as part of this project. What opportunities for training and professional development has the project provided?An undergraduate student who worked on the project was trained in the processes of semen collection, evaluation and data collection. 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?Further studies will be conducted to evaluate cooling of extended ram semen using a variety of storage containers and and cooling times.

Impacts
What was accomplished under these goals? This study was designed to evaluate the quality of extended hair sheep ram semen stored as a liquid at 5°C. St Croix White (STX; n =6) and Dorper x STX (DRPX, n = 5) rams were collected weekly for 3 wk using estrus ewes fitted with an intravaginal collection vial. Semen was kept at 32°C during transport to the lab and during processing. Semen was evaluated for percent motility (MOT), viability (LIVE) by using eosin-nigrosin stain and concentration by using a hemocytometer counting chamber. Semen was extended to a final concentration of 250 x 106/mL in a one-step dilution with a skim UHT milk extender with 10% egg yolk by volume and packaged into 0.5 mL straws. Straws were stored in a styrofoam box in a refrigerator at 5°C for 96 h, or in an Equitainer® set up using the manufacturer's instructions, for 24 h at which time they were transferred to the styrofoam box in the refrigerator for 72 h. Semen was evaluated for MOT and LIVE at -1, 0, 24, 48 72 and 96 h relative to cooling. Semen traits were analyzed using GLM of SAS for repeated measures with ram, time, breed and cooling method in the model. There was no difference (P > 0.10) in MOT or LIVE over time between breeds or cooling method. One STX ram did not produce any samples that survived extension; one STX and four DRPX rams produced at least one sample that did not survive extension and all were removed from analysis. The MOT decreased (P < 0.0001) from 81.7 ± 2.9 % at -1 h to 52.2 ± 2.9% at 96 h. The LIVE decreased (P < 0.0001) from 83.1 ± 3.6% at -1 h to 50.4 ± 3.6% at 96 h. These results show that ram semen stored as a liquid at 5°C can maintain motility and viability for 96 h.

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience: Nothing Reported Changes/Problems:A category 5 hurricane in September 2017 (Maria) severely damaged cattle working facilities and pasture fences. Most of the year has been spent cleaning up debris, making repairs and trying to conduct basic herd management with limited working facilities. Additionally, employees suffered damage to their homes and needed time away from work to deal with that. 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? Due to damage from category 5 hurricane in September 2017 no data was collected on this project. Time has been spent in recovery and clean up of research facilities. Basic animal management was continued.

Publications