Comprehensive Laboratory Meeting Notes: Molecular Research, Clinical Procedures, and Long-Term Strategic Goals

Academic Survival Theory: A core principle shared for success in academia is to "fly under the radar." - This approach implies remaining largely invisible regarding administrative politics to avoid unnecessary duties or public speaking requirements. - The primary benefit Cited is the ability to focus on research and laboratory work without distraction. - The speaker notes an upcoming meeting with the President of the university, acknowledging it as "good news" despite not knowing the specific agenda.

University Cash Handling: High priority is placed on depositing university funds immediately with Elvira. - The speaker expresses a strong personal dislike for being responsible for "university cash" due to the liability and oversight involved.
Animal Sales: Discussion regarding the sale of three animals (rams) to Rams. - Financial details: Money for two animals was received; the third may receive funding from another source.

cDNA Synthesis and RNA Quality: - Caroline reported a poor cDNA run despite good RNA quality. - The target gene for the run was GAP. - There is an urgent need for a new cDNA kit as current supplies for PCR are high, but cDNA-specific kits are depleted.
Laboratory Supply Shortages: - Pipette Tips: Immediate need for $20\,\mu{l}$ and $200\,\mu{l}$ filtered tips. - Non-filtered vs. Filtered: There are current supplies of $200$ non-filtered tips, but filtered ones are required for current protocols. Existing tips do not fit the pipette with the green tip. - Parafilm: The lab is currently out of Parafilm, and a request was made to purchase a dedicated stock for the lab.
Antibody Homology Analysis: - Comparison was conducted between conjugated and human TIM 4 antibodies. - Findings: The conjugated version showed higher homology. - A sheep-specific antibody was also mentioned as a third comparison point. - Caroline is tasked with sending the exact percent homology data to the supervisor.

Current Trial Status: The group discussed a system approach involving $3$ control animals and $3$ treated animals. - These animals were maintained through at least $5$ days/treatments before being euthanized for tissue harvesting. - Confirmation is needed to see if the Kupffer cell approach or the "system approach" is functioning as intended.
T-Cell and Obesity Research: Discussion centered on a specific paper regarding CD4 positive T-cells and "obesity memory." - Phenomena: In individuals with extreme obesity who lose weight, CD4 positive T-cells were found surrounding and inside fat cells. - Mechanism: The cytokine profile of these cells likely facilitates regaining weight; the cells appear to sense nutrient status. - Significance: These cells are also vital for pregnancy recognition. The lab is considering if they are "barking up the wrong tree" with Kupffer cells and should instead investigate these T-cells or a combination of both.
Future Methodology: Liver Biopsies: - The lab proposes isolating immune cells directly from liver biopsies in sheep. - Plan: Perform biopsies, allow healing, and then perform flow cytometry on the isolated cells. - Economic Incentive: This allows the research to continue while still being able to sell pregnant ewes later (referred to as a "money bag").

Vasectomy Procedures: - Tyler requested vasectomies for $3$ more billies (goats) and $1$ ram for the upcoming breeding season. - Semen Testing: Scheduled for next Wednesday with Eric to confirm the success of previous procedures using a scope to check volume and cell presence.
Surgical Schedule: - May 19 (Tuesday): Identified as the primary window for surgery (vasectomies). - July 22: Start date for the first round of AI (Artificial Insemination). The vasectomized males must be cleared (electro-ejaculated) and ready by this time. - July 14: Target date for having the males "squared away" and sterile for at least $8$ weeks.
Institutional Approvals: There was a discussion regarding IACUC approval for performing repeat surgeries on the same animal if a vasectomy fails. Concerns were raised about whether a second surgery on the same site would be permitted.
Anesthesia Logistics: Review of the gas machine operation and filling requirements for surgeries with Dr. Ashley.

Course Concept: Proposal for an Applied Repro Course or Applied AI Core to be launched next spring. - Curriculum: Includes semen handling, cell counting, BSEs (Breeding Soundness Exams), dilution, freezing straws, and AI techniques.
Financial Investment: Realizing the course requires a significant initial investment, estimated near $\$250,000$ . - This includes buying cows and feed (which Tyler offered to float). - The risk involves market fluctuations when selling the animals later.

Grant Deadline: The grant must be completed by July. This necessitates having "preliminary data" and successful cDNA results by early summer.
Arturo’s Test Plate Protocol: - Treatment applied to a test plate, with readings scheduled for $05:30$ and $06:00$ . - Setting: Alimar blue setting on the reader. - Reading Intervals: $15$ , $30$ , $60$ , $120$ , and $240$ minutes.
Quina’s Data Management: - RNA work is ongoing. - Data Cleaning: Removing data points with values less than $3$ to improve summary aesthetics.
Critical Questions for Grants: - Immunofluorescence (IF): Visual proof of the system working. - PCR: Molecular proof of efficacy. - CBC Data: Complete Blood Count metrics. - Progesterone ( $P4$ ): Checking if steroid metabolism was altered. Results are pending review by Dr. Gifford.

High-Expense Items: Arturo noted the need for more plates (approximately $25$ special plates).
General Supplies: - Gloves: Large quantities are needed for upcoming surgeries. - Cell Culture: T75 flasks ( $20$ needed), T125 flasks, and freezing vials. - Surgical Prep: Sutures, needles, packs, gauze, and sterile drapes/towels.

Question: What happens if a semen test shows a vasectomy didn't work? - Response: The team would have to consult Dr. Ashley regarding the possibility of a "re-surgery," though this may conflict with IACUC protocols.
Question: Are there trends in Artificial Insemination equipment for sheep? - Response: It is currently very difficult to find Transcervical AI guns. Most practitioners use Laparoscopic AI or the "Splash" method.
Anecdote regarding AI Equipment: The speaker recounted a time in the Navajo Nation (Arizona) where they only had a half $cc$ gun but quarter $cc$ straws. They improvised a plunger using a small marker flag from road construction, which fit the straw perfectly.