HOW DOES THE MILK OLIGOSACCHARIDE COMPOSITION AND MICROBIOME IMPACT THE SUCCESSION OF THE RUMINAL MICROBIAL POPULATION IN BEEF CATTLE?

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1012273
• Project Start Date: Mar 15, 2017
• Project End Date: Sep 30, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NEW MEXICO STATE UNIVERSITY
1620 STANDLEY DR ACADEMIC RESH A RM 110
LAS CRUCES,NM 88003-1239

Performing Department
Animal & Range Sciences

Non Technical Summary
The composition of the milk from the dam and forage availability and type likely play a role in the time line for rumen development. An area gaining attention in human studies is the oligosaccharide composition of human breast milk. In mammalian species the milk of the dam provides complete nutrition for the offspring during the early phases of development. The paradigm that the mammary gland and milk are devoid of microbial populations is currently being challenged. Additionally, mammalian milk seems to possess bioactive compounds, energy and nutrients that directly influence the development of young mammals. Among the bioactive compounds found in milk are immune substances such as immunoglobulins, cytokines, and chemokines, growth factors, hormones, and antibodies. There are also specific peptides, lactoferrin, whey proteins, oligosaccharides and bacteria. This is an area emerging area of research in regards to human infants however it is largely unexplored in ruminant systems. Therefore, the contribution of these components to animal health and productivity is unknown particularly in nutrient stressed systems in livestock.We intend to investigate these ideas using beef cattle on differing planes of nutrition.

Animal Health Component

40%

Research Effort Categories

Basic

20%

Applied

40%

Developmental

40%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
302	3320	1103	100%

Knowledge Area
302 - Nutrient Utilization in Animals;

Subject Of Investigation
3320 - Meat, beef cattle;

Field Of Science
1103 - Other microbiology;

Keywords

beef cattle

oligosaccharides

Goals / Objectives
Complex and diverse microbial communities mediate the cycling of nutrients within ruminants. The structure of this complex microbial community is shaped by the highly variable physical, chemical, and predatory environment, and potentially by genetic factors of the host. In turn, the microbial community regulates the environment and nutrient availability including the release of energy to the host in the form of volatile fatty acids (Church; 1993.). The diversity of the enteric microbial community is both ecologically and biochemically important. For example, it has been shown that diet can influence the community and functional contrast within the rumen microbial community (Fernando et al., 2010; Fernando et al., 2007). This suggests the microbial diversity and abundance crucially influences nutrient cycling and when inorganic nutrients and carbon are made available for host consumption. With the projection of global demand for meat and milk to double by 2050 (WHO, 2002), identifying microbial populations and factors influence the composition of the microbial population could increase nutrient availability from the rumen will help improve the quality and efficiency of meat production in beef cattle towards meeting this future demand. In mammals, it seems that the gut microbial population is established by successive waves of microbial populations within the community until a stable population is established by time of weaning (Fernando et al., 2010). The establishment of a gut microbial community is influenced by multiple factors, including diet, environment, and host genotype (Benson et al., 2010; Carlisle et al., 2012). Recent beef cattle research has revealed an association between rumen microbial populations and feed efficiency. Feed efficiency is important to the beef cattle industry, especially considering the rapid rise of feed costs. However, feed efficiency is also a complex trait that is difficult and expensive to measure directly. Alternative ways of identifying efficient animals for genetic selection (e.g., rumen microbial profiling) or directly improving feed efficiency (e.g., influencing the early rumen microbiota) are needed for trait advancement.

Project Methods
OBJECTIVES:Objective 1: Characterize milk oligosaccharide and microbiome composition of cows, sheep and goats.PROCEDURES: Milk will be collected from sheep, cattle and goats to assess the oligosaccharide composition. Milk will be collected aseptically every week during lactation and analyzed using FT-ICR MS (Ruhaak and Lebrilla, 2012) for oligosaccharide profile. In addition to oligosaccharide profiles, milk will also be analyzed for components such as protein and fat and microbiome composition by shotgun metagenomics by a commercial laboratory. The number of animals needed for each treatment was determined using calculations with a CV of 2%, a difference of 3%, a P-value of 0.05, and power of 95 (Martin et al., 1987). Number of observations needed per species is 15.Objective 2: Determine the impact of milk oligosaccharide and milk microbiome composition and functionality of microbial populations in the digestive tract of beef calves. PROCEDURES: A total of 40 gestating cows will be used to accomplish this objective. The treatments will be nutrient restricted and unrestricted and will be applied during the last trimester of pregnancy. Cows assigned to the nutrient restricted treatment will be nutrient restricted by 80% of NRC requirements and unrestricted cows will be fed at 100% of nutrient requirements. Samples will be collected from cows and calves at d 7, 60, 120, and 180 in relation to calf birthdate. Samples to be collected include milk and digestive tract swabs and tissue from the rumen, small intestine and large intestine. Samples will be analyzed for microbiome and virome composition as well as oligosaccharide, immunoglobulins, cytokines, and chemokines. Microbiome and virome will be analyzed using shotgun metagenome techniques (Fernando et al., 2007). Immunoglobulin, cytokine and chemokines will be detected using Western blot and quantitative PCR (Jost et al., 2015). Oligosaccharides in the milk will be detected using FT-ICR MS (Ruhaak and Lebrilla, 2012). Five calves from each treatment will be selected to be sampled at each time point. Digestive tract tissue and microbiome swab samples will be collected surgically by a veterinarian under anesthesia so no animals will be sacrificed for this work. The number of animals needed for each treatment was determined using calculations with a CV of 2%, a difference of 3%, a P-value of 0.05, and power of 95 (Martin et al., 1987). Number of observations needed per treatment is 20.

Progress 03/15/17 to 09/30/21

Outputs
Target Audience:The target audience is academic researchers and cattle producers. Changes/Problems:There has been challenges and difficulties related to analyzing milk samples for oligosaccharides. My student was supposed to travel to UC Davis to conduct the analysis but the lab is not accepting students outside of their program. We have developed and validated this analytical protocol at NMSU. 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? This project has 2 graduate students and 2 undergraduates invovled in the research. We are still working through the research and data collection. Covid 19 has slowed the progress of this project due to commercial labs being closed or work delayed due to staffing shortages.

Publications

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

Outputs
Target Audience:Research was presented virutally at the Western Section for American Society of Animal Science. Changes/Problems:The analysis methodology for the milk oligosaccharides has been challenging and we have been working to validate the method and increase the robustness of the procedure. We had issues beginning in June to procure reagents due to shortages that related in delays in research productivity. What opportunities for training and professional development has the project provided?This project has 2 graduate students and 2 undergraduates invovled in the research. How have the results been disseminated to communities of interest?The results are still in progress and have been slowed due to Covid 19 restrictions. What do you plan to do during the next reporting period to accomplish the goals?We are striving to have samples analyzed and ready to presentation at scientific meetings this summer.

Impacts
What was accomplished under these goals? Methodology for analysis and quantification of oligosaccharides in milk has been validated for cattle, sheep, and goats. Samples were collected samples from multiple breeds of cattle, from differing planes of nutritionand microbial analysis will be conducted.

Publications

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

Outputs
Target Audience:The target audience for this project isanimal scientists and livestock producers with an interest in calf health. For the period under review, three abstracts were presented at national scientific meetings and presentations were given at three field day events. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students involved in this research have attend three scienitfic meetings and have expanded their skill set in analytical chemistry by validating the methodology. 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?During the next reporting cycle, animal studies will be completed now that our analysis method has been validated.

Impacts
What was accomplished under these goals? Methodology for analysis and quantification of oligosaccharides in milk has been validated for cattle, sheep, and goats. Research has been initiated to collect samples from multiple breeds of cattle and microbial analysis will be conducted.

Publications

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

Outputs
Target Audience:The target audience for this project is animal scienctist and livestock producers with an interest in calf health. For the period under review three abstracts were presented at national scientific meetings and presentations were given at three field day events. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students involved in this research have attend three scienitfic meetings and have expanded their skill set in analytical chemistry by validating the methodology. How have the results been disseminated to communities of interest?Results are being complied and summarized for presentation at scientific meetings in Summer 2020. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting cycle animal studies will be completed now that our analysis method has been validated.

Impacts
What was accomplished under these goals? Methodology for analysis and quantification of oligosaccharides in milk has been validated for cattle, sheep, and goats. Research has been initiated to collect samples from multiple breeds of cattle and microbial analysis will be conducted.

Publications

Progress 03/15/17 to 09/30/17

Outputs
Target Audience:Data from this project was presented to the 2017 Congress on Gastrointestinal Function in Chicago as a graduate student poster in the Graduate Student poster competition. The student placed 4th out of 27 posters. The meeting was attended by 225 attendees composed of government agency scientist, university faculty and graduate student, and industry research people. This was an international meeting with poeple from 13 different countries. A separate dataset from this project was also presented at the 2017 Western Section of American Society of Animal Science annual meeting in Fargo, North Dakota. The research was presented as an oral presentation in the graduate student competition. There were 170 attendees composed primarily of university faculty and graduate students from the USA, Mexico, Brazil, and Canada. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has allowed for the training of one graduate student and one undergraduate student. Both students learned new techniques in the laboratory and improved communication skills by presenting research at international and national scientific meetings. How have the results been disseminated to communities of interest?The results have been dissemiated by presentations atinternational and national scientific meetings. Additionally updates have been provided to the advisory board of the Corona Range Livestock Research Center. What do you plan to do during the next reporting period to accomplish the goals?During the next phase the work will be repeated to gain a more robust dataset. Additionally, cow milk will be sampled and characterized for oligosaccharide composition. A graduate and undergraduate student will also be a part of this project.

Impacts
What was accomplished under these goals? Ruminants are typically born with a non-functional rumen devoid of microorganisms. The succession of the microbial population in the rumen from birth to animal maturity is of interest due to the key roles that the rumen microbial population plays in the overall health and productivity of the host animal. There is limited data available regarding the development of bacterial populations in beef calves housed on pastures. We hypothesize calves raised in differing environments will alter rumen bacterial population development. The objective of this study was to investigate the effects of environment on the succession of the bacterial population in the rumen of nursing beef calves over time. Ruminal samples were collected on day 7, 35, 63, 91, 119, 147, and 175 of age via oral lavage from 10 Angus calves in New Mexico (NMANG) and Nebraska (NEANG). The pastures at each location differed by plant species and diet quality. Ruminal fluid samples were used for metagenome analysis of the V1-V3 region of the 16S rRNA gene using the Illumina MiSeq 600 cycle v3 sequencing kit. A total of 122 samples produced 333,068 operational taxonomic units (OTU), after filtering for quality and removing apparent chimeras, for an average of 2,338 for NEANG and 2,936 NMANG. Phyla present differed by (P <0.05) environment within day. Bacteroidetes and Firmicutes were the predominant phyla regardless of environment. Bacteroidetes was greater in NEANG at day 35 while day 175 was greater for NMANG. On day 7 Bacteroidetes was less in NMANG however by day 175 NMANG Bacteroidetes was greatest compared to NEANG. Firmicutes was the predominant phyla for NMANG on day 7 and by day 35 Bacteroidetes became the prevailing phyla. Phylum richness continually increased until day 147 for NEANG, while NMANG richness fluctuated throughout the sampling period. Prevotella, CF321, Fibrobacter, and Campylobacter showed significant population differences on day 7 as NEANG more than doubled NMANG population but showed no differences by day 175. The order Bacteroidales supported the genera data. Butyribibrio was significantly greater in NMANG on day 7 but showed no differences by day 175. Genera richness increased from day 7 to day 175 with NEANG having greater richness throughout sampling days. Results show environmental effects that may be driven by diet quality and composition on the succession of the bacterial population in nursing beef calves. The impact of this research is that producers have options with their cattle that had not been realized. The functionality of the rumen at an early age opens up the opprotunity to early wean calves and enhance the reproductive performance of the cow herd. Increases as small as 10 % can equate to thousand of dollars in revenue to producers.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Smith, K. 2017. Effect of age and location on the establishment of microbiome in range beef calves. MS Thesis. New Mexico State University
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Smith, K., Rosasco, S., Beard, J., Oosthuysen, E., Meyerhoff, B., Scholljegerdes, E. J., & Ivey, S. L. (2017). Change of season impacts ruminal fermentation and microbiome in heifers grazing native range (Vol. 68, pp. 153157). Journal of Animal Science.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Butterfield, K., K. Smith, Rosasco, S., Beard, J., Oosthuysen, E., Meyerhoff, B., Scholljegerdes, E. J., & Ivey, S. L. (2017). Effect of age and location of ruminal fermentation in beef calves (Vol. 68, pp. 10). Journal of Animal Science.
• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Smith, K., Rosasco, S., Beard, J., Oosthuysen, E., Meyerhoff, B., Scholljegerdes, E. J., & Ivey, S. L. (2017). Season impacts ruminal fermentation and microbiome in heifers grazing native range (Vol. 68, pp. 153157). Journal of Animal Science.