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• Published: 18 July 2023

Evidence and reporting standards in N-of-1 medical studies: a systematic review

• Prathiba Natesan Batley   ORCID: orcid.org/0000-0002-5137-792X 1 ,
• Erica B. McClure 2 ,
• Brandy Brewer 1 ,
• Ateka A. Contractor 3 ,
• Nicholas John Batley 4 ,
• Larry Vernon Hedges 5 &
• Stephanie Chin 1  

Translational Psychiatry volume  13 , Article number:  263 ( 2023 ) Cite this article

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N-of-1 trials, a special case of Single Case Experimental Designs (SCEDs), are prominent in clinical medical research and specifically psychiatry due to the growing significance of precision/personalized medicine. It is imperative that these clinical trials be conducted, and their data analyzed, using the highest standards to guard against threats to validity. This systematic review examined publications of medical N-of-1 trials to examine whether they meet (a) the evidence standards and (b) the criteria for demonstrating evidence of a relation between an independent and an outcome variable per the What Works Clearinghouse (WWC) standards for SCEDs. We also examined the appropriateness of the data analytic techniques in the special context of N-of-1 designs. We searched for empirical journal articles that used N-of-1 design and published between 2013 and 2022 in PubMed and Web of Science. Protocols or methodological papers and studies that did not manipulate a medical condition were excluded. We reviewed 115 articles; 4 (3.48%) articles met all WWC evidence standards. Most (99.1%) failed to report an appropriate design-comparable effect size; neither did they report a confidence/credible interval, and 47.9% reported neither the raw data rendering meta-analysis impossible. Most (83.8%) ignored autocorrelation and did not meet distributional assumptions (65.8%). These methodological problems could lead to significantly inaccurate effect sizes. It is necessary to implement stricter guidelines for the clinical conduct and analyses of medical N-of-1 trials. Reporting neither raw data nor design-comparable effect sizes renders meta-analysis impossible and is antithetical to the spirit of open science.

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Introduction.

N-of-1 studies, which are special cases of single case experimental designs (SCEDs), are important in the medical field, where treatment decisions may be made for an individual patient, or where large-scale trials are not always possible or even appropriate such as when treating rare diseases, comorbid conditions, or concurrent therapies [ 1 ]. In fact, n-of-1 trials have been suggested as a valuable scientific method in precision medicine [ 2 ] and are particularly important in the field of psychiatry. Recently, the British Journal of Psychiatry published a special issue focusing on precision medicine and personalized healthcare in psychiatry. The What Works Clearinghouse (WWC [ 3 , 4 ]) standards for SCEDs noted several requirements to increase rigor pertaining to evidence standards and demonstration of treatment effect between the independent and the outcome variable. It is important to note here that the term outcome variable refers to the dependent variable and not a medical outcome such as morbidity, mortality, etc. The purpose of these standards is to address validity concerns in SCEDs. What is unclear is if these important standards have been adopted in medical research. To this end, we conducted a systematic literature review using the PRISMA (Preferred Reporting Items for Systematic Literature Reviews and Meta-analyses) guidelines to address the following aims:

To examine whether N-of-1 trials meet WWC evidence standards; namely, independent variables being systematically manipulated, outcome variables measured systematically over time by more than one assessor, interobserver agreement data being collected in each phase for at least 20% of data points per condition, including 3 or more attempts to demonstrate a treatment effect at three different points in time, and having the number of required data points per case/phase,

To examine whether evidence of a treatment effect is examined in N-of-1 trials per WWC standards (namely, immediacy, consistency, changes in level/trend, and effect sizes), and

To examine the data and methodological characteristics of the studies such as phase lengths, inclusion of autocorrelation, appropriateness of the type of analysis for the data type, sensitivity, and subgroup analyses.

Although the SPENT (SPIRIT extension for N-of-1 trials) checklist [ 5 ] has been developed specifically for n-of-1 protocols, we chose the WWC standards because the former focuses on improving the completeness and transparency of N-of-1 protocols, whereas the latter focuses on addressing threats to validity and reporting guidelines to establish evidence of treatment effect between the independent and the outcome variable. Therefore, the latter speaks more to the validity aspects of N-of-1 trials.

Literature search

Inclusion/exclusion criteria.

The review followed the 2020 PRISMA recommendations [ 6 ] (Supplementary Table 1 ) and guidelines from the Cochrane Collaboration for data extraction and synthesis [ 7 ]. Included studies were peer-reviewed, published in medical journals, examined medical outcomes, used SCED/N-of-1, were empirical articles, and in the English language. Only medical conditions listed in International Classification of Diseases (ICD-10) [ 8 ] or Diagnostic and Statistical Manual of Mental Disorders (DSM-5) [ 9 ] were included in the present study to retain a meaningful scope and align with widely used clinical practices. Online Supplementary Table 1 gives the PRISMA checklist and how they were met for the current study.

Search strategy

The following databases were searched: PubMed and Web of Science. These databases were chosen because these search engines have reproducible search results in different locations and at different times. Exact search terms were: "n-of-1*" OR "N-of-1 trial" OR "N-of-1 design" OR "single case design" OR “single subject design” OR “single case experimental design” AND “drug” OR “therapy” OR “intervention” OR “treatment” in the title, abstract, or keywords. The dates of publication were restricted to between January 1, 2013 and May 3, 2022 for relevance, sufficiency, and feasibility as the WWC Standards for SCEDs were published in 2010 and later in 2013. The search ended on May 3, 2022.

Data management

References and abstracts of articles found from the initial search were downloaded into the reference management software EndNote. Duplicate reference entries were removed. The remaining reference entries were transported to a Google Sheets file by and for independent review by two co-authors (EM and BB) for inclusion criteria. Reliability of electronic search results was established through replication of the electronic search and an inter-rater comparison of the number of identified articles (100% agreement).

Selection process

Two co-authors independently (EM and BB) screened 341 articles (title and article abstract review) to determine eligibility of articles for the current review. From this initial screening, 189 articles were identified as potentially eligible and were subjected to a second screening. The two co-authors then independently reviewed the 189 articles (full text) to ensure their eligibility for this review. Articles that were not empirical work (e.g., protocol, commentary), and articles that were not N-of-1 trials or did not have a medical outcome variable were excluded independently leading to a total of 115 articles that met all inclusion criteria (100% agreement).

Coding process

There were 4 coders. Two were experts in statistical methodology and three were experts in SCEDs. One co-author (EM), as the primary coder, conducted data extraction from 115 eligible articles. To obtain inter-reliability estimates, 30 (26.09%) of the included articles were additionally coded by two other co-authors (BB and SC) through random assignment. Before coding the articles included in the review, researchers calibrated coding reliability by using the coding tool to analyze studies that did not meet the inclusion criteria. Interobserver agreements during calibration were measured at 94.3%. When discussing whether a specific study met an individual indicator, areas of incongruity were discussed until researchers reached consensus. Once reliability above 90% was established, researchers began coding the articles included in the review (coding tool available from first author). Interobserver agreement for all coded articles was measured at 93.1%. Finally, the first author (PNB) recoded all the articles to ensure 100% agreement between the first author and the coding of the other three co-authors.

Risk of bias assessment

as given by the Risk of Bias in Systematic Reviews Tool (ROBIS) ( http://www.bristol.ac.uk/population-health-sciences/projects/robis/ ) is in Table 1 . Additionally, the online Supplementary Table 2 gives the risk of bias in not meeting evidence standards, in reporting treatment effect, and in inappropriate data analysis for each study.

Rating evidence

All studies were N-of-1 studies. According to Oxford center for evidence-based medicine (OCEBM, https://www.cebm.ox.ac.uk/files/levels-of-evidence/cebm-levels-of-evidence-2-1.pdf ) all these studies will be level 3 studies because they manipulate the control arm of a randomized trial (Fig. 1 ).

The number of articles identified, screened, retrieved, assessed, and finally retained. n represents the sample size.

Statistical analysis

Descriptive analysis such as frequency and percentages are reported. Table 2 outlines information on number of studies meeting the WWC [ 3 , 4 ] requirements for meeting evidence standards. Table 3 outlines information on number of studies demonstrating how treatment effect was determined per WWC standards [ 3 , 4 ] (immediacy, changes in level/trend, effect sizes/confidence or credible intervals, consistency, effect sizes), and different methodological characteristics (e.g., type of analysis conducted, whether this was appropriate for the data [if data met the assumptions of the analyses], and whether autocorrelations were included in the models). Additionally, we coded the characteristics of the study such as the number of phases, phase length, type of outcome variable, types of effect sizes, data distribution assumptions met, accounting for carryover effect, intraclass correlation, sensitivity analysis, and subgroup analysis.

As outlined in Table 2 , of the 115 studies, 68 (59.13%) did not identify the type of SCED used. Therefore, we identified these designs. To answer research question 1 about how many studies passed all the WWC criteria: only 4 (3.48%) studies passed all the WWC criteria for meeting evidence standards (see online Supplementary Table 2 ). Specifically, IOA (interobserver agreement) was not collected in each phase for at least 20% of data points per condition for 95.7% of the cases. It is possible that sometimes this is not applicable when the outcome variable is measured using an instrument and not necessarily by observers. However, this was the case for only 3 (2.6%) of the studies. 39.3% of the studies did not include ≥ 3 attempts to demonstrate a treatment effect at three different points in time which is a threat to validity because at least 3 independent demonstrations of treatment effect are required to show that the treatment effect did not happen due to random variation in data. Demonstrating a treatment effect at least 3 times is important in N-of-1 studies because the question of whether the treatment effect is replicable across phases or cases is answered by this demonstration, which has obvious impact on validity. 24.8% of them did not have the number of required data points (3–5) per case/phase. This means that the studies were terribly underpowered. It is impossible to obtain reliable estimates of phase means or worse yet, determine if the treatment effect varied with time.

Regarding research question 2, as outlined in Table 3 , most studies (98.3%) determined change in level between phases to report evidence of treatment effects. Consistency was not investigated by 72.6% of the studies and 38.5% of the studies did not report any effect size. The most reported effect size was an unstandardized mean difference between the phases which ranged from −8 to 100. Further, 60.7% of the studies did not report a confidence/credible interval estimate for effect size. The issue with simply reporting an unstandardized mean difference effect size is that there are no units to understand the metric of the effect size. For instance, an unstandardized mean difference of 3 units would be a significant drop in hemoglobin A1C versus a trivial 3 unit drop in systolic blood pressure.

Regarding question 3, only 6% of the studies determined immediacy which is a requirement for causal evidence in SCEDs. Immediacy informs the researcher as to how immediately a treatment took effect, so it eliminates any other extraneous reason for a change in the outcome variable. Therefore, in the absence of a substantive reason for delay in the treatment influencing the outcome variable, immediacy is paramount. Autocorrelation was not modeled in 83.8% of the studies. Of these, one study reported a statistically impossible autocorrelation value of 2. When not including autocorrelations for autocorrelated data, we are assuming the data are independent of each other and any parametric analysis that is employed would be used on data that violate the basic independence of observation assumption. This could lead to wildly inaccurate estimates. We coded the analysis as not being appropriate for the data if the data type did not meet the distributional assumptions of the type of analysis being conducted (65.8%). Again, this could lead to inaccurate estimates. Meta-analysis can be conducted when authors provide a reliable design-comparable effect size estimate or report the complete dataset which is common practice in SCEDs using a data plot. Only one study (0.9%) included autocorrelation and corrected for small sample size in their computations by reporting a design-comparable effect size, i.e., Hedges’ g [ 10 , 11 ]. 47.9% of the studies did not report raw data to be considered for future meta-analysis. Although several studies included more than one participant, only 15.4% computed and reported intraclass correlation. Intraclass correlation is necessary to be computed because it is the correlation among the scores within the individuals or the ratio of between cluster variance (i.e., the variability between people) to the total variance. That way we know how much of the variance in the outcome variable is due to differences between people and within people. This is also necessary to compute the appropriate effect size.

It is highly concerning that only 4 of the 115 (3.48%) studies met the WWC evidence standards. While it has become the default that the presence or absence of phenomena be accompanied by a measure of its magnitude, it is still unfortunate that this essential practice is not being upheld universally. The most reported mean difference effect size is not scale free and therefore, is difficult to interpret and aggregate across studies in meta-analysis. Other effect sizes were Cohen’s d, rate ratio, incidence ratio, etc. which did not include autocorrelation or correction for small sample size in their computations. These are also not design-comparable because they are within-subject effect sizes that are not computed across participants. Regarding immediacy, there are models developed specifically to determine immediacy and its magnitude that can help strengthen the evidence of effective treatments [ 12 , 13 ]. It would behoove medical N-of-1 researchers to examine these methodologies.

Not including autocorrelation in the statistical model is problematic because we know that SCED data are autocorrelated and not modeling autocorrelation leads to erroneous estimates of effect and inflated Type-I error rates [ 14 , 15 , 16 , 17 , 18 , 19 ]. Estimating autocorrelations with sufficient accuracy for shorter time-series is still in its fledgling stage, but it certainly cannot be ignored. We should also remember that most SCEDs have shorter time-series and/or fewer individuals which implies that violation of distributional assumptions becomes more serious, and results are more erroneous when these are ignored. This drawback is exacerbated by ignoring autocorrelations. Reporting intraclass correlation is important for understanding how similar the subjects were to each other and reporting adequate information such as the raw data or a design-comparable effect sizes is essential for scientific progress through meta-synthesis.

Given the findings, here are our recommendations for policymakers, gatekeepers of research standards, and editors of journals that are interested in ensuring the most robust level of conduct and analysis of N-of-1 trials.

Discuss the implications and set standards based on guidelines for best practices founded not just on research conduct in one discipline, but on interdisciplinary research conduct. Specifically, the social sciences and education/psychology research analyses of SCEDs are conducted based on the standards set forth by the WWC. The SPENT guidelines [ 5 ] focus on completeness and transparency of N-of-1 protocols. However, there is a need to derive the best practices based on both and combine the wealth of knowledge created in both fields.

WWC lays specific standards for meeting evidence standards and reporting treatment effect because the data are shorter time-series, are autocorrelated, and often have few participants. These must be strictly adhered to, especially in medical research which has high stakes impact for the subjects.

Use common terminology to facilitate interdisciplinary research. A classic example is identifying the type of SCED. This would allow us to set clear expectations for conduct of research following the highest standards.

Support more methodological contributions in medical journals particularly with respect to analyzing N-of-1 data.

Emphasize the imperative nature of reporting effect sizes and confidence/credible intervals when statistically analyzing data; in fact, make this a requirement.

Report enough information to facilitate meta-analysis, for that is the ultimate aim of most research. This can be either in the form of raw data or design-comparable effect sizes.

Encourage investigation of the impact of autocorrelations in estimating effect sizes. Require the inclusion of estimating and reporting autocorrelations.

Support more development of innovative and easy-to-use tools for analysis of N-of-1 analyses.

The limitations include including only English language articles, potentially excluding articles that might not have been part of the databases we searched in, human errors in coding (although we had 4 independent coders) and excluding N-of-1 studies that did not use our search terms in their title, abstract, or keywords.

In sum, N-of-1 studies in the medical field are not currently adhering to important standards that guard validity both in their conduct and in their analysis. Neither do they report adequate information to facilitate meta-analytic work. Editorial and research standards must require increased rigor in this experimental design which is still in its nascent stage.

Data availability

All data used in this article are made available through online supplemental Table 2 . This also contains all the variables used for coding and the references.

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This work was funded by the grant from the Institute of Education Sciences R305D220052 to the first author of this manuscript.

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PNB conceptualized the study. EM, BB, SC, and PNB conducted the literature search, coded, and conducted the analyses. PNB, AAC, NJB, and LVH co-wrote the manuscript. EM, BB, and SC assisted with editing and formatting.

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Natesan Batley, P., McClure, E.B., Brewer, B. et al. Evidence and reporting standards in N-of-1 medical studies: a systematic review. Transl Psychiatry 13 , 263 (2023). https://doi.org/10.1038/s41398-023-02562-8

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Case studies, single-subject research, and N of 1 randomized trials: comparisons and contrasts

Affiliation.

• 1 Department of Health Care & Epidemiology, School of Rehabilitation Sciences, The University of British Columbia, Vancouver, Canada.
• PMID: 10088595
• DOI: 10.1097/00002060-199903000-00022

Case studies, single-subject research designs, and N of 1 randomized clinical trials are methods of scientific inquiry applied to an individual or small group of individuals. A case study is a form of descriptive research that seeks to identify explanatory patterns for phenomena and generates hypotheses for future research. Single-subject research designs provide a quasi-experimental approach to investigating causal relationships between independent and dependent variables. They are characterized by repeated measures of an observable and clinically relevant target behavior throughout at least one pretreatment (baseline) and intervention phase. The N of 1 clinical trial is similar to the single-subject research design through its use of repeated measures over time but also borrows principles from the conduct of large, randomized controlled trials. Typically, the N of 1 trial compares a therapeutic procedure with placebo or compares two treatments by administering the two conditions in a predetermined random order. Neither the subject nor the clinician is aware of the treatment condition in any given period of time. All three approaches are relatively easy to integrate into clinical practice and are useful for documenting individualized outcomes and providing evidence in support of rehabilitation interventions.

Publication types

• Double-Blind Method
• Effect Modifier, Epidemiologic
• Guidelines as Topic
• Medical Records*
• Randomized Controlled Trials as Topic / methods*
• Rehabilitation
• Reproducibility of Results
• Research Design / standards*

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N-of-1 study: comparative studies

How to use an N-of-1 study to evaluate your digital health product.

This page is part of a collection of guidance on evaluating digital health products .

N-of-1 studies focus on observing changes in individuals (single cases) over time, in comparison to a group-based design in which outcomes are combined for many participants. N-of-1 design (also known as a single-case design study) is used to understand within-person processes, such as changes in an individual’s engagement with your digital product over a period of time.

What to use it for

Similar to group-based designs, an N-of-1 study can be:

• observational (you record what you see)
• experimental (you compare periods when a digital product or features are provided with periods when they are not provided)

This means it can be used during development (formative or iterative evaluation) to find out how to improve your product. It helps you to explore its more nuanced effects and determine, for example, what factors predict higher usage.

An N-of-1 study can also be used to evaluate the effects of your digital product by providing detailed data on how the effect varies for a single person. Because of this, it can be used to inform the design of a comparative study (see Choose evaluation methods: evaluating digital health products ).

Benefits of N-of-1 studies include:

• they can provide more granular data on changes in an outcome than you would get from combining outcome data across participants
• they can identify individuals a product works for and does not work for
• they generally require fewer participants in comparison to more traditional group-based studies
• in an experimental evaluation, each participant acts as their own control, meaning the design is more sensitive to differences in the effects of the intervention

Drawbacks of N-of-1 studies include:

• they often involve intense data collection that might be a burden for some participants
• you might need expert statistical skills to analyse the data
• there is a risk of missing data because of the repetitive nature of collecting the data – this can make the analysis more complex

How to carry out an N-of-1 study

The most rigorous of this type of study is an N-of-1 trial. In a traditional randomised controlled trial ( RCT ), groups of participants are randomly allocated to an intervention or a control condition. In an N-of-1 trial, individuals are assigned to different options in a randomly-determined order, so they are exposed to the intervention and control on different days of the trial period (called multiple crossovers).

Because a participant experiences the intervention and the control over different times, some interventions are not appropriate for assessing in an N-of-1 trial. For example, if the:

• intervention takes more time than a typical treatment period to produce an effect (slow onset effect)
• intervention produces a change that remains for some time after the intervention stops (carry-over effect)
• health condition is progressing rapidly

This means N-of-1 trials are good for interventions that are reversible (where the effect wanes over a short time), but not appropriate for interventions which have a long-lasting effect.

Between the periods, you can introduce a washout period, in which, to allow the effects of the previous intervention to diminish, participants receive no intervention. This can help with some slow-onset and carry-over effects. Piloting your digital intervention can also help you to identify any slow-onset and carry-over effects, and how significant they might be.

The statistical power (see ‘Statistical power’ in Design your evaluation: evaluating digital health products ) of N-of-1 studies depends on the number of observations rather than the number of participants. N-of-1 designs typically use ecological momentary assessment, which involves frequent data collection at different intervention phases.

An N-of-1 study can include just one person, but typically a series of N-of-1 studies are undertaken. These can either be analysed as separate datasets, or in some cases combined statistically to provide an average effect between participants. If a large number of N-of-1 datasets are combined, then it is possible to identify participant characteristics that are associated with the intervention effect (for example, to identify who the intervention works for and who it does not work for).

The data from an N-of-1 study should be treated as a time series: repeated observations of a particular measurement collected over time. It’s important to note that individual responses tend to be more similar when assessments are carried out close together in time (autocorrelation). For example, when responding to a questionnaire about how you are feeling, your responses given yesterday and tomorrow will usually be more similar to each other than your reponses given a week ago or a week in the future.

You will need to analyse the data using models that account for autocorrelation, either as single cases or combined in a multilevel analysis.

Example: evaluating the effect of goal-setting and self-monitoring on increasing walking

See Sniehotta and others (2012): Testing self-regulation interventions to increase walking using factorial randomized N-of-1 trials .

Researchers wanted to investigate the effectiveness of 2 interventions on walking: goal-setting versus self-monitoring.

They used a factorial N-of-1 trial where they assessed how effective goal-setting and self-monitoring features are in increasing walking. This design has similar principles to group-based factorial RCTs . This was a series of 2 × 2 factorial N-of-1 studies where 10 participants were randomised to either a goal-setting condition or a control and also to either a self-monitoring condition or a control.

In the goal-setting condition, participants were prompted to set themselves a goal to achieve a specific number of steps. The goal-setting control included a goal to consume more fruit and vegetables on that day.

For the self-monitoring condition, participants were given 2 pedometers, one with a visible display and one with a sealed (blinded) display, to monitor their steps.

Participants received a text message prompt each morning, telling them which goal and pedometer they needed to use.

The researchers conducted 10 regression analyses, one per participant, to find out for whom goal-setting and self-monitoring were effective. They controlled for autocorrelation. The study did not have the statistical power to detect interaction effects. Read more about interaction effects in factorial RCTs .

Researchers found that most participants increased their number of steps in both the goal-setting and self-monitoring conditions compared to control days. However, individual analyses showed different effects of the interventions:

• 4 participants significantly increased walking – 2 on self-monitoring days and 2 on goal-setting days
• one participant showed a small decrease in their steps throughout the study

This study showed the variability of the effects of these 2 commonly-used ways to increase activity, suggesting that one size does not fit all.

More information and resources

Dallery and others (2013): Single-case experimental designs to evaluate novel technology-based health interventions . This article discusses how N-of-1 studies can be useful for assessing digital technology.

Agency for Healthcare Research and Quality (2014): Design and implementation of N-of-1 trials: a user’s guide . A comprehensive guide on how to conduct N-of-1 studies.

Kwasnicka and Naughton (2020): N-of-1 methods: A practical guide to exploring trajectories of behaviour change and designing precision behaviour change interventions . This paper provides a guide to analysing N-of-1 data, including how to account for autocorrelations in N-of-1 studies.

Kwasnicka and others (2018): Challenges and solutions for N-of-1 design studies in health psychology . This article outlines the challenges of doing N-of-1 studies and gives solutions for overcoming them.

Examples of N-of-1 studies in digital health

Odineal and others (2020): Effect of mobile device-assisted N-of-1 trial participation on analgesic prescribing for chronic pain: randomized controlled trial . The team conducted an N-of-1 trial and used an app to facilitate the running of the study.

Lee and others (2020): “Asking too much?”: A randomised N-of-1 trial exploring patient preferences and measurement reactivity to frequent use of remote multidimensional pain assessments in children and young people with juvenile idiopathic arthritis . An N-of-1 study to explore the most acceptable way to measure pain levels in children and young people.

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Nitrate contamination in groundwater and its health risk assessment: a case study of Quanzhou, a typical coastal city in Southeast China

• Original Article
• Published: 13 May 2024
• Volume 83 , article number  331 , ( 2024 )

Cite this article

• Zhenghong Li 1 , 2 ,
• Jianfeng Li 1 , 2 ,
• Jin’ou Huang 3 &
• Yasong Li 1 , 2  

Nitrate contamination has become an ecological and health issue in Quanzhou, a typical coastal city in Southeast China. Hydrogeological surveys reveal that NO 3 − is a major factor influencing the groundwater quality in Quanzhou City, Fujian Province, China. To protect public health, this study explored the geographical spatial distribution, contamination level, contamination sources, and noncancer risks of nitrates in the plain area of Quanzhou. Key findings are as follows: (1) The groundwater in Quanzhou’s plain area exhibits a high detection rate and over-limit ratio of NO 3 − –N of 99.3% and 57.86%, respectively. This result suggests that the groundwater in the area has been extensively contaminated by nitrates, with relatively severe nitrate contamination occurring in the Quanzhou Taiwanese Investment Zone, Jinjiang City, and Shishi City; (2) NO 3 − has become a major anion in groundwater in Quanzhou’s plain area, leading to significant geochemical changes in some groundwater. 26.4% of the groundwater samples exhibited a hydrochemical type of nitric acid (also referred to as NO 3 − type water), with X(NO 3 − ) ≥ 25%; (3) The primary nitrate contamination in groundwater in Quanzhou originates from the infiltration of domestic and industrial wastewater or landfill leachate; (4) 42.86%, 43.57%, and 67.14% of the samples posed health risks to adult males, adult females, and children, respectively when they were subjected to the prolonged exposure in a high-concentration nitrate environment. Additionally, the noncancer risks of nitrates principally stem from oral exposure for drinking water.

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Acknowledgements

This work was supported by the China Geological Survey project (Nos. DD20190303, DD20221773, DD20230459) and Zhejiang Provincial Geological Special Fund Project (No. 2023010)

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Li, Z., Li, J., Huang, J. et al. Nitrate contamination in groundwater and its health risk assessment: a case study of Quanzhou, a typical coastal city in Southeast China. Environ Earth Sci 83 , 331 (2024). https://doi.org/10.1007/s12665-024-11608-z

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Importance of multimodal resident education curriculum for general surgeons: perspectives of trainers and trainees

• Jeeyeon Lee 1   na1 ,
• Hyung Jun Kwon 1   na1 ,
• Soo Yeon Park 1 &
• Jin Hyang Jung 1  

BMC Medical Education volume  24 , Article number:  518 ( 2024 ) Cite this article

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Metrics details

Satisfaction should be prioritized to maximize the value of education for trainees. This study was conducted with professors, fellows, and surgical residents in the Department of general surgery (GS) to evaluate the importance of various educational modules to surgical residents.

A questionnaire was administered to professors ( n  = 28), fellows ( n  = 8), and surgical residents ( n  = 14), and the responses of the three groups were compared. Four different categories of educational curricula were considered: instructor-led training, clinical education, self-paced learning, and hands-on training.

The majority of surgeons regarded attending scrubs as the most important educational module in the training of surgical residents. However, while professors identified assisting operators by participating in surgery as the most important, residents assessed the laparoscopic training module with animal models as the most beneficial.

Conclusions

The best educational training course for surgical residents was hands-on training, which would provide them with several opportunities to operate and perform surgical procedures themselves.

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Introduction

Continuous, lifelong medical education should be provided because it is essential to training qualified specialists in every medical field [ 1 ]. Educational periods may be categorized as medical college, postgraduate, and specialist education. The residency training program is the most important part of postgraduate education. However, departments of general surgery (GS) in the United States and South Korea are seeing decreased demand for surgical residencies [ 2 , 3 , 4 ].

The decreasing demand for residencies in GS is critical because it results in a lack of specialists. The reasons for this demand reduction vary and include the fatigue accumulated from emergency surgeries, the deterioration of their quality of life, and the frequent stressful situations caused by handling vital organs [ 5 , 6 , 7 ]. Nevertheless, many physicians continue to choose surgical residency and might feel enthusiastic when they succeed in performing surgery, thus saving patients’ lives and improving patients’ odds of survival. Although a majority of surgical applicants apply with this intent, they experience frustration and often regret their decision when faced with the harsh reality of the field. In light of these problems, surgical residency training should pay attention to the mental and physical well-being of residents. In particular, unlike the trainers’ generation, residents of the twenty-first century prioritize work-life balance, technological proficiency, adaptability to pandemic situations, diversity, and inclusion [ 8 , 9 , 10 , 11 ]. These factors have gained even greater emphasis following the coronavirus pandemic. Given that residents in Korea tend to avoid challenging but essential medical care, motivation becomes even more critical for those applying to GS. Educational programs that fail to consider these specific characteristics may lead to decreased achievement and efficiency.

Experts agree that instructors must consider trainees’ satisfaction to maximize educational impact [ 12 , 13 ]. Each qualified specialist’s training involves numerous educational training modules, which should be accessible and tailored to the demands of surgery. Instructors should determine how to increase educational impact by verifying the degree of satisfaction of surgical residents and evaluating the efficacy of the educational curriculum. Educational programs are generally divided into instructor-led training (ILT) and self-paced learning (SPL), and the program should be structured to balance these two areas equally [ 14 , 15 , 16 , 17 , 18 ]. Moreover, in the education of doctors, each resident must receive not only theoretical education but also more detailed programs related to clinical and technical skills.

In this study, by evaluating the responses of professors, fellows, and surgical residents in a department of GS regarding trainee satisfaction and the importance of educational training modules, we identified the most appropriate educational curriculum for residents to establish better training courses toward cultivating specialists with higher qualifications.

The educational curricula for surgical residents were organized by the Education Committee of the department of surgery at Kyungpook National University Hospital, Daegu, Republic of Korea. The categories of educational curricula were classified into ILT, clinical education, SPL, hands-on training, and detailed training courses (Table  1 , Supplementary Fig.  1 ). ILT is defined as a training and learning program provided by instructors or teachers, whereas SPL is a student-driven learning design [ 18 , 19 , 20 ].

The importance of different categories of resident education was assessed on the basis of a cross-sectional questionnaire proffered via e-mails or text messages. Professors, fellows, and surgical residents responded to the survey. Academic faculty and clinical specialists comprised the group of professors. Fellows were defined as specialists who had completed their residency within the immediately preceding two years. The training system for medical residents in Korea consists of a three-year format, and accordingly, surgical residents consisted of first-year, second-year, and third-year residents following the curriculum of the Korean Surgical Society. All the professors and fellows, who have successfully completed their residency and obtained specialist qualifications, were regarded as educators. And only residents in their first to third years of training were considered as trainees.

The questionnaire comprised nine items, including the level of the surgeons; their self-estimated daily working hours; and the time they estimated to have devoted to the education of residents in a week, excluding routine jobs (Supplementary Table  1 ). The top three important education curricula among the 15 training courses constituting the educational curricula were further classified into the four categories.

Each complete response collected from professors, fellows, and residents was analyzed and presented in the form of bar graphs for convenient comparison.

Sociodemographic characteristics

In total, 50 surgeons responded to the questionnaires. Six of the respondents repeated their submissions because of incomplete forms. The mean ages of the groups of professors ( n  = 28), fellows ( n  = 8), and residents ( n  = 14) were 47.3 years (SD, ± 15.1), 33.3 years (SD, ± 2.3), and 31.0 years (SD, ± 3.3), respectively.

Professors specialized in breast/thyroid (n = 7, 25.0%), colorectal ( n  = 6, 21.4%), upper gastrointestinal ( n  = 5, 17.9%), vascular ( n  = 4, 14.3%), hepato-bilio-pancreatic ( n  = 3, 10.7%), pediatric ( n  = 1, 3.6%), trauma (n = 1, 3.6%), and critical care ( n  = 1, 3.6%) surgeries. Fellows majored in vascular ( n  = 3, 37.5%), colorectal ( n  = 2, 25.0%), upper gastrointestinal ( n  = 1, 12.5%), breast/thyroid ( n  = 1, 12.5%), and critical care ( n  = 1, 12.5%) surgeries. Fourteen residents from the first year ( n  = 2, 14.3%), second year ( n  = 4, 28.6%), and third year ( n  = 8, 57.1%) participated (Supplementary Table  3 ).

Self-estimated daily working time and time devoted to the education of residents in a week.

Professors responded that they worked 8 h ( n  = 8, 28.6%), 10 h ( n  = 13, 46.4%), and more than 12 h ( n  = 7, 25.0%) per day. However, a majority of fellows ( n  = 6, 75.0%) and residents ( n  = 12, 85.7%) responded that they worked for more than 12 h per day. Professors and residents spent approximately 2 h per week on the education of surgical residents, excluding routine jobs (Supplementary Table  2 ).

Most helpful educational category for the training of surgical residents.

Of the four different educational categories, clinical education was regarded as the most helpful educational course for surgical residents by all groups. A marginal majority of professors (by a slight margin) regarded the ILT course as the most helpful to education. While fellows selected clinical education and SPL as the most and least helpful courses, surgical residents identified hands-on training and SPL as the most and least helpful courses, respectively (Fig.  1 ).

Importance ratings of four major educational categories for surgical residency training indicate that clinical education was considered the most important (*) curriculum overall. However, each group, including professors, fellows, and residents, judged different curricula to be the most important

Top three important educational curricula for surgical residents

The identification of the three most important curricula among the 15 training courses covered in the four categories was performed by way of a featured survey question. As shown in Table  2 , each group selected attending scrubs as the most important program among the three most important courses.

Importance of training courses in the ILT category for the training of surgical residents

All the surgeons selected general lectures by professors as the most important curriculum in the ILT category for the training of surgical residents. Notably, fellows and residents considered this curriculum the most important course to a greater extent than professors did. By contrast, professors and residents considered residents’ participation in regular in-hospital conferences the least important in resident training, whereas fellows considered the Surgical Grand Rounds as less important than other curricula (Fig.  2 ).

Importance ratings of instructor-led training (ILT) courses for surgical residency training indicate that general lectures were considered the most important (*) part of the curriculum overall. All groups consistently judged general lectures to be the most important educational program

Importance of the training course in the clinical education category of surgical residents’ training.

All groups responded that attending scrubs was the most important training method in clinical education. While the professor and resident groups ranked attending inpatient and outpatient clinics as second and third in importance, respectively, the fellow group rated the clinics with a similar degree of importance in clinical education (Fig.  3 ).

Importance ratings of courses within the clinical education category for surgical resident training indicate that attending scrubs are considered the most important, both overall and by each group

Importance of the training course in the SPL category for the training of surgical residents

All groups considered the regular journal club the most important training course in the SPL category. While the professor and fellow groups considered the manual for surgical residents less important than other curricula, residents regarded it as the third-most important course. Surgical residents considered collecting data and writing articles the least important (Fig.  4 ).

Importance ratings of self-paced learning (SPL) courses for surgical residency training indicate that resident journal club was considered the most important (*) part of the curriculum overall. And all groups consistently judged resident journal club to be the most important educational program

Importance of the training course in the hands-on training category for surgical residents’ training

In the hands-on category, the dry-lab laparoscopy training module within the hospital was deemed by professors as the most important training course for surgical residents, whereas the laparoscopic training module in the professional education center with animal models was perceived as most important by fellows and residents (Fig.  5 ).

Importance rating of courses within the hands-on training category for the training of surgical residents. While the professor group judged that dry lab laparoscopy training module was the most important (*), the fellow and resident groups judged the animal laparoscopy training module to be more important

This study revealed that trainers and trainees differ in their criteria for evaluating the importance of educational components, with surgical residents particularly interested in hands-on training. Halsted emphasized the significance of clinical and practical training in the education of American surgeons [ 21 ]. Similarly, current GS professors and residents place a high value on practical and technical education. However, stringent ethical standards present challenges for residents practicing directly on cadavers or patients [ 22 , 23 , 24 , 25 , 26 ]. Consequently, professors typically offer thorough theoretical training before introducing practical skills to mitigate potential complications. The contemporary environment is causing divergent approaches between trainers and trainees. If an education system that closely simulates human conditions is developed, and a system where trainees can freely receive education within ethical boundaries is established, it could become the most efficient approach to education.

A series of educational courses must be provided to medical students, interns, residents, and fellows to ensure medical specialists’ optimal training. Although these training courses may be delivered at various levels depending on the knowledge and needs of trainees, instructors have predominantly solely developed the ILT curriculum. However, the actual effectiveness of training courses may differ from the instructors’ expectations; furthermore, considering the satisfaction of the trainees while planning the courses could improve the learning experience [ 12 , 13 , 27 ].

When designing curricula, it is crucial to consider that surgical residents apply for residencies because they are interested in surgery and are aspired to be surgeons. Some professors focus on teaching academic theory, while others contend that mastering the art of thesis writing is the most critical aspect of education. Nonetheless, the most basic desire of surgeons must be considered. In our current study, we found that the opinions of professors, fellows, and residents regarding the most important training courses for surgical residents were similar.

While the group of professors believed that watching many standardized surgeries is the most important training course needed by surgical residents to participate and assist in surgeries, residents considered having practical operating experience as the most important. Therefore, residents regarded the laparoscopic training module in the professional institute with animal models as the most important curriculum in the training of surgical residents. The responses from the group of fellows, who had completed their residency within the past two years, revealed a trend that intermediates between the views of professors and residents. Given their transitioning status from learners to educators, their opinions are considered very important. In fact, these opinions represented a compromise between the perspectives of professors and residents in their roles as educators.

Each of the different learning methodologies of ILT and SPL has advantages and disadvantages [ 15 , 16 , 17 ]. ILT offers detailed materials and immediate feedback but is limited by structured schedules and instructor variability. SPL allows flexibility in timing and location with the use of open-source materials, enabling repeated review of content, though it lacks the immediate feedback of ILT and may require more time for comprehension. Ultimately, trainees benefit most from actively engaging with the educational material and their learning process.

Although this study focused on resident education, only 14 residents were actually included which is a limitation of this study. However, unfortunately, this is the real-world situation in South Korea. Many doctors are avoiding essential medical services, such as internal medicine, GS, obstetrics and gynecology, and pediatrics. Instead, a majority prefer departments like plastic surgery, ophthalmology, and dermatology, which focus on cosmetic procedures or enhancing the quality of life. This is the reason why, despite being a fairly large-scale national university hospital, the number of residents was small. This situation underlines the importance of further improving the quality of education.

The responses in this study can help standardize the appropriate educational programs for surgical residents in GS departments. Based on these research findings, the authors' institution has decided to enhance technical education with theoretical support. It is currently developing and implementing education on ultrasound and biopsy techniques, laparoscopic bowel anastomosis, and robotic surgery. We established close cooperation with other departments so that in addition to surgical skills training, we could directly experience and learn other departments' techniques (intubation, ventilator manipulation, CPR, and so on.). Various colleagues and companies are supporting this training, viewing it as an investment in the future generation of surgeons. As demonstrated in the authors' study, it is necessary to verify and evaluate the educational programs of each institution for their rationality and efficiency. This approach will contribute to the training of better surgeons by enabling residents to engage more actively in the curriculum.

The educational impact of training materials and methods can be maximized when surgical residents engage in the preferred training resources that provide them with satisfaction. Therefore, the best educational training course for surgical residents would include providing them with many opportunities to operate and perform surgical procedures themselves. Although written materials and theories remain important, the effect of education is enhanced when the surgical residents’ satisfaction is increased through the provision of practical learning opportunities.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author, JHJ, upon reasonable request.

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Acknowledgements

The authors thank to Byeongju Kang, Byunghyuk Yu, Deokbi Hwang, Gyu-Seog Choi, Ho Yong Park, Ho Young Chung, Hye Jin Kim, Hyung Kee Kim, Ji Yeon Park, Jin Ho Chung, Jin Young Park, Jun Seok Park, Ki Bum Park, Kyoung Hoon Lim, Oh Kyoung Kwon, Sang Geol Kim, Seung Huh, Seung Soo Lee, Seung Ho Song, Sung-Min Lee, Wan Wook Kim, Woo Sung Yun, Yang Soo Lim, Yoon Jin Hwang, Dong-hee Na, Heejung Keum, Hui je Jeong, Hyeon Ju Kim, Jae Yeong Yang, Min Hye Jeong, Sue hyun Park, Young Hye Kim, Eunji Kim, Gunwoo Kim, Gyoyeong Chu, Hakyung Yoon, Hannah Kim, Hoyeon Kwon, Hyunjae Kang, Jeong Eon Suh, Ji Hyun Jung, Junhyun Park, Seokhyun Wi, Sujin Kang, Suyeong Hwang, Taewan Ku, all of whom participated in the survey of this study.

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Jeeyeon Lee and Hyung Jun Kwon contributed equally to this work.

Authors and Affiliations

Department of Surgery, Kyungpook National University Chilgok Hospital, Hoguk-Ro 807, Buk-Gu, 41404, Daegu, Republic of Korea

Jeeyeon Lee, Hyung Jun Kwon, Soo Yeon Park & Jin Hyang Jung

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Guarantor of the integrity of the study: JL, Study concept: JL, JHJ, Study design: JL, JHJ, Definition of intellectual content: JL, SYP, HJK, Literature research: SYP, HJK, Data acquisition: JL, JHJ, HJK, Data analysis: JL, SYP, Manuscript preparation: JL, JHJ, Manuscript review: JHJ, Manuscript editing: JHJ, JL. All authors have read and approved the manuscript.

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Correspondence to Jin Hyang Jung .

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This study was approved by the Institutional Review Board Committee of Kyungpook National University Chilgok Hospital, Daegu, Republic of Korea (KNUCH 2022–07-024–001). Informed consent was obtained from all subjects.

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Lee, J., Kwon, H.J., Park, S.Y. et al. Importance of multimodal resident education curriculum for general surgeons: perspectives of trainers and trainees. BMC Med Educ 24 , 518 (2024). https://doi.org/10.1186/s12909-024-05515-x

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A Peek Inside the Brains of ‘Super-Agers’

New research explores why some octogenarians have exceptional memories.

By Dana G. Smith

When it comes to aging, we tend to assume that cognition gets worse as we get older. Our thoughts may slow down or become confused, or we may start to forget things, like the name of our high school English teacher or what we meant to buy at the grocery store.

But that’s not the case for everyone.

For a little over a decade, scientists have been studying a subset of people they call “super-agers.” These individuals are age 80 and up, but they have the memory ability of a person 20 to 30 years younger.

Most research on aging and memory focuses on the other side of the equation — people who develop dementia in their later years. But, “if we’re constantly talking about what’s going wrong in aging, it’s not capturing the full spectrum of what’s happening in the older adult population,” said Emily Rogalski, a professor of neurology at the University of Chicago, who published one of the first studies on super-agers in 2012.

A paper published Monday in the Journal of Neuroscience helps shed light on what’s so special about the brains of super-agers. The biggest takeaway, in combination with a companion study that came out last year on the same group of individuals, is that their brains have less atrophy than their peers’ do.

The research was conducted on 119 octogenarians from Spain: 64 super-agers and 55 older adults with normal memory abilities for their age. The participants completed multiple tests assessing their memory, motor and verbal skills; underwent brain scans and blood draws; and answered questions about their lifestyle and behaviors.

The scientists found that the super-agers had more volume in areas of the brain important for memory, most notably the hippocampus and entorhinal cortex. They also had better preserved connectivity between regions in the front of the brain that are involved in cognition. Both the super-agers and the control group showed minimal signs of Alzheimer’s disease in their brains.

“By having two groups that have low levels of Alzheimer’s markers, but striking cognitive differences and striking differences in their brain, then we’re really speaking to a resistance to age-related decline,” said Dr. Bryan Strange, a professor of clinical neuroscience at the Polytechnic University of Madrid, who led the studies.

These findings are backed up by Dr. Rogalski’s research , initially conducted when she was at Northwestern University, which showed that super-agers’ brains looked more like 50- or 60-year-olds’ brains than their 80-year-old peers. When followed over several years, the super-agers’ brains atrophied at a slower rate than average.

No precise numbers exist on how many super-agers there are among us, but Dr. Rogalski said they’re “relatively rare,” noting that “far less than 10 percent” of the people she sees end up meeting the criteria.

But when you meet a super-ager, you know it, Dr. Strange said. “They are really quite energetic people, you can see. Motivated, on the ball, elderly individuals.”

Experts don’t know how someone becomes a super-ager, though there were a few differences in health and lifestyle behaviors between the two groups in the Spanish study. Most notably, the super-agers had slightly better physical health, both in terms of blood pressure and glucose metabolism, and they performed better on a test of mobility . The super-agers didn’t report doing more exercise at their current age than the typical older adults, but they were more active in middle age. They also reported better mental health .

But overall, Dr. Strange said, there were a lot of similarities between the super-agers and the regular agers. “There are a lot of things that are not particularly striking about them,” he said. And, he added, “we see some surprising omissions, things that you would expect to be associated with super-agers that weren’t really there.” For example, there were no differences between the groups in terms of their diets, the amount of sleep they got, their professional backgrounds or their alcohol and tobacco use.

The behaviors of some of the Chicago super-agers were similarly a surprise. Some exercised regularly, but some never had; some stuck to a Mediterranean diet, others subsisted off TV dinners; and a few of them still smoked cigarettes. However, one consistency among the group was that they tended to have strong social relationships , Dr. Rogalski said.

“In an ideal world, you’d find out that, like, all the super-agers, you know, ate six tomatoes every day and that was the key,” said Tessa Harrison, an assistant project scientist at the University of California, Berkeley, who collaborated with Dr. Rogalski on the first Chicago super-ager study.

Instead, Dr. Harrison continued, super-agers probably have “some sort of lucky predisposition or some resistance mechanism in the brain that’s on the molecular level that we don’t understand yet,” possibly related to their genes.

While there isn’t a recipe for becoming a super-ager, scientists do know that, in general , eating healthily, staying physically active, getting enough sleep and maintaining social connections are important for healthy brain aging.

Dana G. Smith is a Times reporter covering personal health, particularly aging and brain health. More about Dana G. Smith

A Guide to Aging Well

Looking to grow old gracefully we can help..

The “car key conversation,” when it’s time for an aging driver to hit the brakes, can be painful for families to navigate . Experts say there are ways to have it with empathy and care.

Calorie restriction and intermittent fasting both increase longevity in animals, aging experts say. Here’s what that means for you .

Researchers are investigating how our biology changes as we grow older — and whether there are ways to stop it .

You need more than strength to age well — you also need power. Here’s how to measure how much power you have  and here’s how to increase yours .

Ignore the hyperbaric chambers and infrared light: These are the evidence-backed secrets to aging well .

Your body’s need for fuel shifts as you get older. Your eating habits should shift , too.

People who think positively about getting older often live longer, healthier lives. These tips can help you reconsider your perspective .

The ALIRT Revelation: Exposing $1.3 Billion LOC Fraud in the Insurance Industry

T he insurance and reinsurance industry operates on principles of trust and financial stability. Central to this stability is the concept of collateral, which provides security for policyholders and reinsurers alike. However, recent revelations have brought to light a troubling trend: the potential misuse of letters of credit (LOCs) in collateralized reinsurance transactions. The ALIRT revelation has cast a shadow over the integrity of the industry, raising questions about due diligence, transparency, and accountability.

The ALIRT Investigation: Peeling Back the Layers

In a bid to shed light on this complex issue, ALIRT Insurance Research embarked on a comprehensive investigation. Their findings were startling. Through an analysis of US insurer filings, ALIRT uncovered a staggering $1.3 billion worth of LOCs from China Construction Bank (CCB). This significant exposure immediately set off alarm bells, particularly due to suspicions of fraudulent activities associated with transactions facilitated by Vesttoo, a collateralized reinsurance platform.

Identifying Potential Fraud: Navigating a Gray Area

One of the central challenges in this investigation by ALIRT is distinguishing between legitimate transactions and fraudulent ones. While some insurers have taken proactive measures to replace potentially fraudulent LOCs, the full extent of the problem remains elusive. ALIRT’s analysis underscores the complexity of the situation, highlighting the shared responsibility of all parties involved in these transactions. The blurred lines between legitimate business dealings and fraudulent activities underscore the need for enhanced oversight and vigilance within the industry.

HOA’s Entanglement: A Case Study in Vulnerability

One insurer that found itself ensnared in the collateral conundrum is Homeowners of America Insurance Company (HOA), owned by insurtech Porch Group. With $300 million in CCB LOCs, representing a significant portion of its policyholder surplus, HOA’s predicament serves as a stark reminder of the pervasive nature of the issue within the insurance industry. The repercussions of this entanglement extend beyond financial losses, encompassing reputational damage and eroded trust among stakeholders.

Fronting Specialists Under Scrutiny: Exposing Vulnerabilities

Fronting specialists, such as Clear Blue Insurance Company, featured prominently in ALIRT’s findings, with substantial exposure to CCB LOCs. The prevalence of these specialists in the use of LOCs underscores the need for heightened scrutiny and due diligence in collateralized reinsurance transactions. The concentration of risk among a select group of players raises concerns about systemic vulnerabilities within the industry and the potential for cascading impacts in the event of a widespread failure.

Global Implications: A Ripple Effect Across Borders

The reverberations of the Vesttoo linked collateral fraud saga extend far beyond the borders of the United States. With the involvement of other banks, including Standard Chartered and Santander, the global insurance and reinsurance landscape faces unprecedented challenges. The interconnected nature of the industry means that no market is immune to the fallout from these revelations. Lloyd’s of London, a cornerstone of the insurance market, is not exempt from these issues, further underscoring the need for a coordinated and collaborative response at the international level.

The Call for Transparency and Accountability: Rebuilding Trust

Amidst the uncertainty and complexity surrounding the Vesttoo-linked collateral fraud saga, one thing remains abundantly clear: the urgent need for greater transparency and accountability within the insurance and reinsurance industry. Regulatory bodies, insurers, and reinsurers must join forces to strengthen oversight mechanisms, enhance due diligence practices, and restore confidence in the integrity of the market. Only through concerted efforts can we rebuild trust and fortify the foundations upon which the insurance industry is built.

Questioning Due Diligence: Lessons Learned

The Vesttoo-linked collateral fraud saga serves as a sobering reminder of the inherent challenges of navigating a complex and rapidly evolving industry landscape. ALIRT’s astute observation raises fundamental questions about the efficacy of due diligence processes. How could such widespread fraud evade multiple levels of scrutiny? The answer lies in a collective failure to prioritize diligence over expediency, highlighting the need for a cultural shift towards a more robust and vigilant approach to risk management.

In conclusion, the Vesttoo linked collateral fraud saga represents a watershed moment for the insurance and reinsurance industry. It serves as a wake-up call, compelling stakeholders to confront systemic vulnerabilities, fortify risk management practices, and reaffirm their commitment to integrity and transparency. By learning from the lessons of the past and embracing a culture of continuous improvement, the industry can emerge stronger, more resilient, and better equipped to navigate the challenges of an ever-evolving landscape.

• Open access
• Published: 07 May 2024

Effects of improved amino acid balance diet on lysine mammary utilization, whole body protein turnover and muscle protein breakdown on lactating sows

• Sai Zhang 1 , 2 ,
• Juan C. Marini 3 ,
• Vengai Mavangira 4 ,
• Andrew Claude 4 ,
• Julie Moore 1 ,
• Mahmoud A. Mohammad 3 &
• Nathalie L. Trottier 1 , 5  

Journal of Animal Science and Biotechnology volume  15 , Article number:  65 ( 2024 ) Cite this article

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The study objective was to test the hypothesis that low crude protein (CP) diet with crystalline amino acids (CAA) supplementation improves Lys utilization efficiency for milk production and reduces protein turnover and muscle protein breakdown. Eighteen lactating multiparous Yorkshire sows were allotted to 1 of 2 isocaloric diets (10.80 MJ/kg net energy): control (CON; 19.24% CP) and reduced CP with “optimal” AA profile (OPT; 14.00% CP). Sow body weight and backfat were recorded on d 1 and 21 of lactation and piglets were weighed on d 1, 14, 18, and 21 of lactation. Between d 14 and 18, a subset of 9 sows (CON = 4, OPT = 5) was infused with a mixed solution of 3-[methyl- 2 H 3 ]histidine (bolus injection) and [ 13 C]bicarbonate (priming dose) first, then a constant 2-h [ 13 C]bicarbonate infusion followed by a 6-h primed constant [1- 13 C]lysine infusion. Serial blood and milk sampling were performed to determine plasma and milk Lys enrichment, Lys oxidation rate, whole body protein turnover, and muscle protein breakdown.

Over the 21-d lactation period, compared to CON, sows fed OPT had greater litter growth rate ( P < 0.05). Compared to CON, sows fed OPT had greater efficiency of Lys ( P < 0.05), Lys mammary flux ( P < 0.01) and whole-body protein turnover efficiency ( P < 0.05). Compared to CON, sows fed OPT tended to have lower whole body protein breakdown rate ( P = 0.069). Muscle protein breakdown rate did not differ between OPT and CON ( P = 0.197).

Feeding an improved AA balance diet increased efficiency of Lys and reduced whole-body protein turnover and protein breakdown. These results imply that the lower maternal N retention observed in lactating sows fed improved AA balance diets in previous studies may be a result of greater partitioning of AA towards milk rather than greater body protein breakdown.

The increasing availability of crystalline amino acid (CAA) at competitive costs relative to protein ingredients allows for reduction of excessive dietary nitrogen (N) and improving AA balance [ 1 ]. Several studies have shown that improving dietary AA balance in lactating sows leads to greater milk casein yield [ 2 , 3 ] and utilization efficiency of N and essential amino acid (EAA) [ 4 , 5 ] while dramatically mitigating N losses and ammonia emissions to the environment [ 2 ]. Lysine efficiency values previously reported [ 4 , 5 , 6 ] were estimated based on lean mass change during lactation. This approach yielded similar efficiency estimates for Val based on isotopic method [ 7 ]. Lysine utilization efficiency values in lactation using an isotopic approach have not been reported.

The increased apparent AA efficiency may be at the expense of sow body weight (BW) loss and reduced maternal N retention whereby partitioning of dietary AA and energy towards the mammary glands appears to be favoured [ 3 , 5 , 8 ]. Preserving maternal N pool during the lactation period is important since maternal body protein and lipid loss can affect subsequent production performance. Loss in performances may include delayed estrus [ 9 ], reduction of piglet birth weight and litter uniformity [ 10 , 11 ], and prolonged interval from weaning to successful pregnancy [ 12 ], thus compromising the overall life span production efficiency. It is unknown whether the reduced maternal N retention previously reported [ 3 , 5 ] in sows fed an improved AA balance diet was a result of greater maternal body protein breakdown. In addition, whole body and muscle protein breakdown rates in lactating sows are unknown and such values are critical to assess at a mechanistic level the impact of improved dietary AA balance on body protein dynamic. Isotope technique allows for better mechanistic understanding of protein dynamics, including protein turnover rate, AA flux and muscle protein breakdown in humans [ 13 ] and animals [ 14 ].

We hypothesized that low CP diet with improved AA balance would increase milk yield through improving efficiency of Lys for milk and increasing maternal body and muscle protein breakdown. The objectives were to (1) measure whole body protein dynamics and (2) estimate Lys utilization efficiency for milk synthesis.

Materials and methods

Dietary treatments.

The NRC model [ 6 ] was used to estimate requirements for AA, net energy (NE), calcium (Ca) and phosphorus (P). The requirements were predicted based on the following parameters: sow BW of 210 kg after parturition, parity number of 2 and above, average daily intake of 6 kg/d, litter size of 10, piglet average daily gain (ADG) of 280 g/d over a 21-d lactation period, and an ambient temperature of 20 °C. The model predicted a minimum sow BW loss of 7.5 kg and the protein to lipid ratio in the model was adjusted to the minimum allowable value of near zero. The model predicted SID Lys requirement of 0.90% and NE requirement of 2,580 kcal/kg.

A control diet (CON) was formulated using corn and soybean meal as the only sources of Lys to meet the SID Lys requirement (0.90%) and consequently contained 19.24% CP and a SID Val concentration 0.77% which was near the NRC (2012) requirement of 0.79%. All other EAA SID contents were in excess relative to NRC (2012). The SID AA values of feed ingredients were referred to NRC (2012). A second diet was formulated to improve AA balance [ 5 ], and referred to as optimal diet (OPT) throughout the manuscript. Fermentable fiber was high in CON due to high content of soybean meal with 24.88% fermentable fiber [ 6 ]. Thus, the same fiber source (soy hulls) was supplemented in OPT, and levels of fermentable fiber were consistent between CON and OPT. Ingredients and calculated nutrient composition of CON and OPT diets are presented in Table 1 . Analyzed total (hydrolysate) and free AA concentrations are presented in Table 2 , in order to verify the precision of diet formulation. The analyzed N concentration corresponded to a CP% of 18.44 compared to a calculated value of 19.24% CP. Therefore, the analyzed CP concentration value is used in the heading for the remainder of tables.

Animals and feeding

The study was conducted at the Michigan State University Swine Teaching and Research Center. A total of 18 purebred multiparous (parity 2+) Yorkshire sows were moved to conventional farrowing crates between d 105 and 107 of gestation, grouped by parity, and randomly assigned to 1 of 2 dietary treatments within parity groups (CON, n = 9; OPT, n = 9). The study was conducted over 3 blocks of time, with 6, 6, and 5 sows in each block, respectively. One sow in CON from block 3 was removed due to poor feed intake that was deemed unrelated to the dietary treatments. Sows were adapted to the experimental diets (2.2 kg/d) 4 to 6 d before the expected farrowing date. Following farrowing, sows feed allowance progressively increased from 1.88 kg/d on d 1 to 7.44 kg/d at d 21, according to the NRC model [ 6 ], with a targeted ADFI of 6.0 kg/d over the 21-d lactation period. Feed was provided daily in 3 equal meals (0700, 1300, and 1900) with feed intake and refusal recorded daily before the morning meal. On infusion days (between d 14 and 18), the 0700 and 1300 meals were divided into 6 aliquots fed every 2 h from 0700 to 1700. Water was freely accessible to sows and piglets. Litters were aimed to be standardized to 11 piglets within the first 24 h after farrowing with the objective of weaning 10 piglets per sow. Injection of iron and surgical castration of male were conducted on d 1 and 7, respectively, according to the institutional research farm protocol. No creep feed was supplied to the piglets. Body weight and backfat thickness [ 5 ] of sows were recorded on d 1 and 21, and litter weights were recorded on d 1, 14, 18 and 21. Milk yield was estimated for peak lactation (between d 14 and 18) [ 5 ]. Prediction equation for milk yield during peak lactation is as follows [ 15 ]: \(\mathrm{Daily}\;\mathrm{milk}\;\mathrm{yield}\;(\text{g}/\text{d})=\mathrm{littersize}\times(582+1.168\times\text{ADG}+0.00425\times\text{ADG}^2)\)

Bilateral ear vein catheterization

A subset of 10 sows (5 sows per treatment) was used for the catheterization and infusion protocol. An ear vein catheter was placed in each ear, with one ear serving as the infusion line and the other as the sampling line. For the length of the catheterization procedure, piglets were removed and transferred to an empty adjacent stall with a heat lamp. The sows were restrained with a rope snare and remained in their farrowing stall where sedation was induced. For sedation, Telazol was reconstituted with 2.5 mL of 100 mg/mL ketamine and 2.5 mL of 100 mg/mL xylazine to a volume of 5 mL. This sedative mixture was administered i.m. in the brachiocephalicus muscle approximately 6 cm caudal to the ear, at a dosage of 0.1 mL/4.537 kg body weight. Sows were carefully assisted to facilitate laying in ventral recumbence. Sedation lasted for 45 to 60 min. The depth of anesthesia was monitored by the degree of muscle relaxation and respiration rate (i.e., 10 to 25 breaths/min).

The entire dorsal surface of both ears was prepared for aseptic placement of the ear vein catheters. The skin was scrubbed gently with 10% betadine solution followed with 70% isopropyl alcohol. The hair covering the skin area caudal to the ear and dorsal to the neck was clipped using a professional clipper to ensure a good adhesion of veterinary adhesive tape to the skin (described below).

A pre-cut 61-cm, round tip, medical grade microbore intravascular tubing (1.65 mm o.d., 1.02 mm i.d.) with hydromer coating (Access Technology Corp., Skokie, IL, USA) was prefilled at the time of catheterization with heparinized saline (30 IU/mL) before insertion. A hand tourniquet was applied at the base of the ear to distend the medial and lateral branches of the auricular vein. Either vein was used for catheterization. A short-term stylet catheter (14G, 5.08 cm, Safety IV catheter; B. Braun Melsungen AG, Germany) was inserted into the vein with the needle bevel facing up. Upon appearance of blood, the vein was gently occluded, and the needle rotated 180° to angle the bevel facing down. While holding the needle in place, the stylet catheter was gently pushed into the vein through the needle. Once the stylet was in place, the needle was removed, and the intravascular tubing was inserted through the stylet and pushed for approximately 30 cm caudally to reach the external jugular vein, and the catheter verified for patency at this point. Small sections of tape (5.1 cm wide, ZONAS® porous tape, Johnson & Johnson Consumer Companies, Inc., Skillman, NJ, USA) were affixed to the remaining section of intravascular tubing and sutured to the skin to secure the tubing in place. The stylet catheter was also sutured (Monocryl, CP-1, 36 mm, 1/2c; Ethicon Inc., USA) to the skin at the point of entry. Gauze was placed over each sutured sites and held in position by wrapping the ear with elastic adhesive tape. A connector was used to join the intravascular tubing to a long tubing extension (approximately 120 cm). A blunt-end needle adapter with an adaptor injection cap and a male luer lock was placed onto the distal end of the tubing extension. The same vein catheterization procedure was done on the other ear. A final layer of elastic adhesive tape (7.5 cm wide, 3M veterinary adhesive tape) was used to wrap each ear into a gently folded cone shape and to affix extension tubing directly onto the clipped skin surface. The extension tubing ran from the ears to the dorsal region of the neck, caudally to the ears and cranial to the shoulders and the free end (approximately 100 cm) rolled up and placed in a handmade denim protective pouch mounted on 4.0-cm thick foam material. The pouch was kept in place by gluing the foam directly onto the skin with Livestock ID Tag Cement (W.J. Ruscoe Company, Akron, OH, USA). Catheters were verified for patency once more and the lines were filled with sterile saline, coiled, and placed in the pouch until used for infusion and blood sampling. The entire procedure was done following sterile techniques and lasted 45 to 90 min per sow. As soon as sows were able to stand, 15-cm wide elastic bandage (Novation ® , Hartmann USA, Inc., Rock Hill, SC, USA) was wrapped over the pouch and around the neck and thorax in at least 3 layers in the shape of a life vest (crisscross) to protect the pouch. Thereafter, the catheters were verified for patency and flushed with sterilized heparinized saline (30 IU/mL) twice per day.

Catheters were removed after all infusions and blood sampling were completed (blood sampling lasted for 3 d for 3MH; Fig. 1 ). The elastic bandaging was removed, and the elastic adhesive tape was carefully pulled to expose the sutures. The sutures were cut with small surgical scissors, the catheters were gently pulled out of the ear veins, and pressure was applied over the insertion sites to accelerate coagulation. The remaining adhesive tape was then carefully removed, and the pouch was freed from the foam which remained on the sow. Rectal temperature was recorded from the day of catheterization and for 3 d following removal of catheters.

Plasma isotopic enrichment of 3-[methyl- 2 H 3 ]histidine following 3-[methyl- 2 H 3 ]histidine bolus infusion during peak lactation (between d 14 and 18) for sows fed control (CON; 18.4% CP; n = 4) and optimal (OPT; 14.0% CP; n = 4) diets. Plasma isotopic enrichment of 3-[methyl- 2 H 3 ]histidine differed between diets ( P < 0.001) and time points ( P < 0.001), with no interaction between diet and time ( P = 0.894). Standard error of the mean (SEM) = 0.214

Preparation of isotope solutions

Tracers were weighed, dissolved in saline and the solution sterilized by filtration through Millipore Steriflip filters (0.22 μm). For each sow, the following stock solutions were prepared: 3-[methyl- 2 H 3 ]histidine (183 μmol in 20 mL saline for bolus injection), [ 13 C]bicarbonate (368 μmol in 20 mL saline for prime and 736 μmol in 30 mL saline for 2-h infusion), and [1- 13 C]lysine (1.28 mmol in 30 mL saline for prime and 9.00 mmol in 60 mL saline for 6-h infusion). The bolus dose of 3-[methyl- 2 H 3 ]histidine (3MH) was calculated based on 20% pool size of 3MH in sows [ 16 , 17 ]. The infusion rate of [1- 13 C]lysine was calculated based on average flux of lysine (25 mmol/h) in lactating sows [ 7 ] with the aim of 2% enrichment. The priming dose of [1- 13 C]lysine was aiming for 1.5 mmol (1 h of infusion), and 1.28 mmol was the actual amount according to weight balance.

The solution of [ 13 C]bicarbonate was freshly prepared to minimize loss of 13 CO 2 . Specifically, [ 13 C]bicarbonate was weighed and dissolved in 20-mL 3-[methyl- 2 H 3 ]histidine solution in the morning of infusion day (Fig. 2 ). The 3-[methyl- 2 H 3 ]histidine (3MH) was used to estimate muscle protein breakdown, and the [ 13 C]bicarbonate was used to prime the CO 2 pool to accelerate the estimation of lysine oxidation rate. The primed-constant infusion of [1- 13 C]lysine was used to estimate lysine utilization by the mammary gland and the lysine flux in the whole body.

Timeline of isotope infusion and sampling (infusion day was within d 18–21)

Infusion protocol

The timeline for infusion is presented in Fig. 2 . Actual infusion day varied between d 14 and 18 due to real time patency of catheter. For lysine balance (Table 3 ) and body protein kinetics (Table 6 ), the actual infusion days were 17.0 ± 1.0 for CON and 17.0 ± 1.4 for OPT. For 3MH kinetics, the actual infusion days were 17.3 ± 1.0 for CON and 16.8 ± 1.5 for OPT. Pumps (Genie Touch TM , Kent Scientific Corp, Torrington, CT, USA) and syringes were placed on a large and stable plastic board laid above the farrowing stall. Following the priming dose, the infusion line was immediately attached to the syringe mounted to the pump to begin the constant infusion. The sampling line was coiled and stored in the pouch until used for blood sampling.

The mixed 20 mL saline solution containing 3-[methyl- 2 H 3 ]histidine (183 μmol) and [ 13 C]bicarbonate (368 μmol) was given through the infusion line as a bolus injection. The [ 13 C]bicarbonate in this infusate was used as a priming dose. After bolus injection, a constant 2-h infusion of [ 13 C]bicarbonate (368 μmol/h) began. The 2-h [ 13 C]bicarbonate infusion was followed by a 6-h primed constant [1- 13 C]lysine infusion (1.50 mmol/h) (Fig. 2 ).

Blood sampling

The timeline for blood sampling is presented in Fig. 2 . For analysis of plasma 3-[methyl- 2 H 3 ]histidine concentrations and estimation of muscle protein breakdown rate, blood samples were collected through the sampling line at 0 (immediately after termination of the bolus infusion), 5, 10, 15, 30 and 45 min and 1, 2, 3, 4, 5, 6, 7, 8, 24, 34, 48, 58 and 72 h post bolus infusion. Blood samples (0.5 mL) were transferred into 500-μL BD microtainer tubes (K 2 EDTA) and centrifuged (1,500 × g at 4 °C for 5 min). The plasma was extracted and stored in 1.5-mL microcentrifuge tubes at −20 °C until analysis.

For analysis of plasma [1- 13 C]lysine concentrations and estimation of whole-body Lys flux, blood samples (0.5 mL) were collected prior to infusion for background enrichment and at 1, 2, 3, 4, 5 and 6 h from the start of [1- 13 C]lysine infusion (Fig. 2 ).

For analysis of blood CO 2 concentrations, blood samples (2 mL) were collected prior to [ 13 C]bicarbonate-prime infusion for background, and at 1, 2, 3, 4, 5, 6, 7 and 8 h following the prime infusion. Blood samples were injected into evacuated vacutainer tubes (Becton Dickinson, Plymouth, UK) previously prepared with 2 mL of phosphoric acid, immediately mixed, and cooled to room temperature. The CO 2 was then transferred from evacuated vacutainers to Exetainer tubes (Labco Breath Tube, UK) by using pure nitrogen gas as medium until analysis.

Milk sampling protocol

The timeline for milk sampling is presented in Fig. 2 . Milk samples were taken between d 14 and 18 during the infusion protocol. Milk was sampled before infusion for background enrichment, and at 1, 2, 3, 4, 5 and 6 h of primed constant infusion of Lys.

For each milk sampling period, piglets were separated from the sows for 1 h in an empty adjacent farrowing crate with no access to water, and sows were administered 1 mL of oxytocin (20 IU/mL oxytocin, sodium chloride 0.9% w/v, and chlorobutanol 0.5% w/v, VetTek, Blue Springs, MO, USA) through the sampling catheter immediately after blood sampling. The catheter was rinsed with 2 mL of saline solution to ensure oxytocin reached the blood circulation. A total of 30-mL milk was manually collected across all glands and stored in 2 separate 15-mL tubes (polypropylene centrifuge tubes with screw cap, Denville Scientific, Swedesboro, NJ, USA). Piglets were immediately returned to sows to complete nursing and empty the mammary glands. Piglets were removed after nursing and kept separate from the sow until the next milk sampling time, 1 h later.

Isotope analysis

Plasma and milk [1- 13 C]lysine and 3-[methyl- 2 H 3 ]histidine (after acid hydrolysis) were determined as their dansyl derivatives by HESI LC-MS as previously described [ 18 ]. The following m/z transitions were monitored: 613→379 and 614→380 for [1- 13 C]lysine and 403→124 and 406→127 for 3-[methyl- 2 H 3 ]histidine. Determination of blood 13 CO 2 enrichment was performed by IRMS (Delta+XL IRMS coupled with GasBench-II peripheral device, Thermo-Quest Finnigan, Bremen, Germany) as previously described [ 19 ].

Nutrient analysis

Feed samples were analyzed for gross energy (GE) by bomb calorimetry (Parr Instrument Inc., Moline, IL, USA). Dry matter and N in feed samples were analyzed as previously described [ 5 ]. Dietary AA analysis [AOAC Official Method 982.30 E (a,b,c), 45.3.05, 2006] was performed by the Agricultural Experiment Station Chemical Laboratories (University of Missouri-Columbia, Columbia, MO, USA) as outlined in previous reports [ 5 ].

Calculations

The following assumptions were made during calculation:

Priming dose of isotope was assumed to mix with pool instantly.

The appearance of unlabeled bicarbonate was constant during the time of primed-constant infusion of bicarbonate (2 h) and that of [1- 13 C]lysine (6 h).

[1- 13 C]lysine cannot be synthesized once 1-carbon was lost to CO 2 , thus rate of lysine decarboxylation represented rate of lysine breakdown.

Kinetics of plasma lysine was an indicator of kinetics of whole body protein.

The indicator AA (lysine) was assumed to be oxidised for maintenance or incorporated into milk protein without other metabolic pathway.

Lysine oxidation

The enrichment of CO 2 during the period of primed-constant infusion of [ 13 C]bicarbonate was calculated as follows (Eq. 1 ):

Where “infusion \({\text{rate}}_{\text{H}{}^{13}\text{CO}_3^-}\) ” represents the infusion rate (368 μmol/h) of [ 13 C]bicarbonate, and “ \(\text{Ra}_{\text{HCO}_{3}^-}\) ” represents the rate of appearance of unlabeled bicarbonate (baseline) in the body.

The enrichment of CO 2 during the period of primed-constant infusion of [1- 13 C]lysine was calculated as follows (Eq. 2 ):

Where “ \({\text{Ra}}_{\text{H}{}^{13}\text{CO}_3^-}\) ” represents the rate of appearance of labeled bicarbonate from [1- 13 C]lysine oxidation, and “ \(\text{Ra}_{\text{HCO}_{3}^-}\) ” represents the rate of appearance of unlabeled bicarbonate (baseline) in the body as in Eq. 1 .

The enrichment of lysine during the period of primed-constant infusion of [1- 13 C]lysine was calculated as follows (Eq. 3 ):

Where Ra Lys represents the rate of appearance of unlabeled lysine in the body.

Lysine oxidation was estimated as follows (Eq. 4 ):

Whole body protein breakdown and synthesis

Whole body protein breakdown (PB) and synthesis (PS) were mirrored by Lys dynamics (Table 3 and Fig. 3 ).

Isotopic enrichment of [1- 13 C]lysine in plasma (panel a ) and milk (panel b ) over 6 h during peak lactation (d 14 to 18) for sows fed control (CON; 18.4% CP; n = 3) and optimal (OPT; 14.0% CP; n = 5) diets

The PB and PS were calculated as follows (Eqs. 5 and 6 ):

Where 146.19 g/mol is the molar weight of isotopic Lys, 6.74% is the average weight percentage of Lys in the sow’s body protein [ 6 ].

The average milk protein concentration of 5.16% was used [ 6 ]. Milk yield was estimated according to Theil et al. [ 15 ].

Muscle protein breakdown

Data was expressed as tracer to tracee ratio (TTR). Multiexponential models were fitted to the data (Eq. 10 and Fig. 1 ). Residual inspection and pseudo-R 2 were used to determine the most parsimonious model that best fitted the data from each individual sow. Area under the curve (AUC; TTR•h) was calculated using the parameters from the multiexponential equation (Eq. 11 and Table 4 ) and 3MH rate of appearance (Ra; μmol/kg/h) was calculated by dividing the dose administered (μmol/kg) by AUC (Eq. 12 and Table 4 ). Half-life (h) was determined using the rate constant corresponding to the tail of the curve (Eq. 13 , Table 4 , and Fig. 1 ).

Muscle protein breakdown rate (%/d) was calculated as follows (Eq. 14 ):

Where total protein bound 3MH pool = muscle protein mass (g) × 3.8742 μmol 3MH/g protein and muscle protein mass = 8.21% × sow BW (kg) [ 20 ].

Muscle protein breakdown (g/d), was calculated as follows (Eq. 15 ):

Efficiency of lysine for lactation

Lysine utilization efficiency for lactation was calculated as follows (Eq. 16 ):

Statistical analysis

Data were confirmed for homogeneity of residual variance and normality of residuals by Mixed Procedures and Univariate Procedures of SAS 9.4 (SAS Inst. Inc., Cary, NC, USA) before ANOVA analysis (Mixed model procedures).

For the lysine balance and protein kinetic estimation, two sows from CON were removed from the data set. In one case, both ear vein catheters lost patency at the time of infusion, and in the other case, one of the 2 ear vein catheters lost patency. The latter sow, however, was used for the estimation of muscle protein breakdown, which only required one catheter. Therefore, the number of sows for the lysine balance data and protein kinetic estimation were 5 and 3 for OPT and CON, respectively.

For the analysis of lysine enrichment in plasma and milk, the following model was used:

The Enrichment of lysine depended on the fixed effects of diet ( OPT vs. CON ) , and sampling hour, with hour included as repeated measurement. The random effects included block and individual sow . The interactive effect of diet × hour was also included .

For the analysis of lysine balance, body protein breakdown and synthesis, and muscle protein breakdown rate, identified as “Response”, the following model was used:

The Response depended on the fixed effects of diet ( OPT vs. CON ) . The random effects included block and individual sow .

Differences between treatments were declared at P < 0.05 and tendencies at P ≤ 0.1.

Lactation performance

Lactation performance during the 21-d period and milk yield and nutrient concentrations between d 14 and 18 are presented in Table 5 . Sow initial BW and ADFI did not differ between OPT and CON diets. Litter growth rate of sows fed OPT diet was greater than those fed CON diet ( P < 0.05).

Lysine balance and efficiency of utilization

Lysine balance values are presented in Table 3 . The SID Lys intake, Lys oxidation, flux and Lys associated with protein synthesis did not differ between OPT and CON diets (Table 3 ). Compared to sows fed CON, those fed OPT had greater efficiency of Lys (0.62 vs. 0.50; P < 0.05) and tended to have a lower ( P = 0.069) released Lys associated with protein breakdown.

Whole body protein synthesis, whole body protein breakdown and fractional muscle protein breakdown

Whole body protein breakdown rate and synthesis rate tended to be lower ( P = 0.069 and P = 0.109, respectively) and protein turnover efficiency (synthesis: breakdown) tended to be greater ( P = 0.060) in sows fed OPT compared to those fed CON (Table 6 ). Whole body protein net synthesis (i.e., whole body protein synthesis − whole body protein breakdown) did not differ between OPT and CON diets.

For estimation of muscle protein breakdown rate, an additional sow in OPT treatment lost patency of both catheters, therefore the number of sows was 4 in each of the treatment. A 3-exponential model best fitted the 3MH decaying curve (Fig. 1 ) and pseudo- R 2 were > 0.995. Muscle protein breakdown rate and fractional muscle protein breakdown rate (%) did not differ ( P = 0.197) between sows fed OPT and CON diets (4.84% and 5.59%, respectively) (Table 6 ).

Enrichment of lysine

Lysine enrichment in plasma (panel a) and milk (panel b) is presented in Fig. 3 . The lysine enrichment in plasma did not differ between diets and time. Lysine enrichment in milk was lower ( P < 0.01) in sows fed OPT compared to sows fed CON diets and differed over time ( P < 0.01). There was no interaction between diets and time.

fDynamics of 3-[methyl- 2 H 3 ]histidine

Plasma isotopic enrichment of 3-MH following 3-MH bolus infusion is presented in Fig. 1 , and relevant dynamic parameters are presented in Table 4 . Plasma isotopic enrichment of 3-MH of sows fed CON was lower ( P < 0.001) than that for OPT diet. Time effects of 3-MH were significant ( P < 0.001) in both treatments of CON and OPT, and no interaction effect between diet and time ( P = 0.894) was detected.

Previous studies showed that lactating sows fed low CP diets with CAA supplementation had greater milk casein yield [ 2 , 3 ], and utilization efficiency of N and EAA [ 4 , 5 ]. The improvement of milk yield however was at the expense of sow BW and maternal N retention [ 3 , 5 ]. Zhang et al. [ 8 ] suggested that feeding diets with improved AA balance triggered nutrient repartitioning to milk at the expense of maternal adipose tissue rather than protein tissue. Maternal body fat loss affects subsequent reproductive performance and compromises the overall production efficiency during the sow’s life span [ 21 ]. Therefore, commercial implementation of diets with aggressing reduction in CP with CAA supplementation to achieve improved AA balance will not only depend on their impact on lactation performance and production efficiency but also on ensuring that long-term maternal body protein and lipid reserves are not compromised.

The mechanisms behind the reduced maternal N retention in sows fed improved AA balance diets reported in earlier studies [ 4 , 5 ] are unclear. Reduced maternal body protein synthesis, greater body protein breakdown, or a combination of thereof can dictate maternal N balance during lactation. In this study however, BW and backfat change during lactation did not differ between OPT and CON sows. Of note, sows fed OPT had no change in BW with a small loss in backfat while sows fed CON gained 5.5 kg with no change in backfat. Body protein kinetics in this study (Table 6 ) dictated that whole body protein net synthesis (whole body protein synthesis − whole body protein breakdown) of sows fed CON and OPT were close (1,041.72 vs. 1,022.90 g/d), but note that whole body protein net synthesis of lactating sows included milk protein yield and maternal protein deposition. Milk protein yield was greater in OPT than CON as mirrored by litter growth rate (Table 5 ). Consequently, maternal protein deposition was greater in CON than OPT which aligns with the observation that body weight increased in sows fed CON while there was no change of body weight in sows fed OPT (Table 5 ). In addition, increased milk production in sows fed OPT suggest that OPT diet led sows to partition more dietary nutrient towards milk than maternal reserves, in other words, sows fed OPT were more motivated to produce milk even at the expense of maternal deposition.

This study used Lys as representative AA of body protein to analyze whole body protein turnover. In essence, Lys flux in the blood was contributed by dietary Lys intake and Lys released by body protein breakdown, and free Lys in the blood could be directed to either Lys oxidation or Lys incorporation into body protein (Fig. 4 ). Thus, body protein breakdown and synthesis could be estimated by measuring Lys flux in blood and Lys oxidation. The carbon dioxide released by Lys oxidation remains in the blood bicarbonate pool and mixed with carbon dioxide from other substrate oxidation (Fig. 5 ). By priming the bicarbonate pool, the baseline production rate of carbon dioxide can be estimated based on bicarbonate enrichment and constant infusion rate of labeled bicarbonate during prime-constant infusion of bicarbonate (Eq. 1 ). The release of labeled carbon dioxide due to labeled Lys oxidation was proportional to the baseline production rate of carbon dioxide according to enrichment of bicarbonate during prime-constant infusion of Lys (Eqs. 2 and  3 ).

Diagram of lysine balance in lactating sows at fed state

Representation of a two-pool model to estimate lysine oxidation

Milk protein synthesis represents the difference between whole body protein synthesis and breakdown, assuming that maternal protein retention is close to zero, since maternal tissue mobilization is majorly comprised of body fat rather than body protein [ 5 , 8 ]. According to this assumption, milk protein output rate measured by isotopic technique (Eq. 8 ) was 1,023 to 1,042 g/d, which aligns well with a previous study where 957 g/d milk protein synthesis was reported using a N balance approach [ 5 ]. When compared to traditional method where milk protein synthesis is the product of milk yield and milk protein concentration (645 to 675 g/d; Eq. 9 ), the isotopic-predicted milk protein synthesis (1,023 to 1,042 g/d) appears overestimated. Guan et al. [ 7 ] reported milk protein synthesis of 575 g/d as the net balance between sow whole mammary protein synthesis (975 g/d) and breakdown rate (400 g/d), corroborating the values reported here (645–675 g/d) using the traditional method. Nitrogen balance techniques tend to overestimate actual nitrogen retention [ 22 , 23 ], as observed herein with the isotope technique (Eq. 8 ). Note that the estimated muscle protein breakdown rate according to 3MH method in this study was 960 to 1,261 g/d (Eq. 15 ), which was greater than the protein breakdown rate (650 to 1,051 g/d; Eq. 8 ) based on the Lys flux. On the other hand, milk protein synthesis rate per metabolic BW (BW 0.75 ) were 10.25 and 9.85 g/d/kg 0.75 for OPT and CON, respectively in this study, supporting a previously reported value of 11.57 g/d/kg 0.75 (using Val as representative AA) [ 24 ]. Thus, overestimation of milk protein synthesis (Eq. 8 ) was majorly attributed to an underestimation of protein breakdown rather than overestimation of protein synthesis. The underestimation of body protein breakdown according to Lys flux (Eq. 5 ) may be partially due to the tendency to overestimate feed intake [ 23 ], although feed waste was minimized in this study. Nevertheless, it is also important to note that estimated muscle protein breakdown (15.4 to 17.8 μmol/kg/d; Eq. 15 ; Table 6 ) and fractional breakdown (4.84–5.59%/d; Eq. 14 ; Table 6 ) in this study was greater than those reported for lactating gilt (3.4%/d, 12.0 μmol/kg/d) using the same 3MH method [ 17 ]. It is speculated that the multiparous lactating sow may mobilize body protein more readily compared to the lactating gilt.

In this study, milk protein yield of lactating sows fed OPT diet did not differ from those fed control diet neither when an isotopic method nor the traditional method were used. Although there was no difference between whole body protein synthesis and breakdown, the absolute values of protein synthesis and breakdown were both lower in sows fed OPT diet compared to CON diet, suggesting less whole-body protein turnover in sows fed the OPT diet. In support of this view, previous studies also showed a decreased protein breakdown rate reflected by lower urea nitrogen output when sows were fed reduced protein diets [ 3 , 5 ]. The biological process of protein turnover is energetically costly [ 1 , 25 ]. Zhang et al. [ 5 ] reported feeding sows with an improved AA balance diet was associated with higher energy efficiency, lending support to the current observation.

The Lys efficiency for sows fed different levels of dietary protein based on the NRC [ 6 ] approach was previously determined [ 4 , 5 ], with greater efficiency values (0.68 and 0.66, respectively) found during peak lactation (d 14–18) in sows fed low CP diets balanced with CAA. Herein, greater Lys utilization efficiency values, determined using a different approach, were also found in sows fed OPT (0.62) compared to CON (0.50). The estimation of Lys utilization efficiency was based on Lys balance parameters, i.e., Lys flux in blood, SID Lys intake and Lys oxidation (Fig. 4 ), and the assumption that net protein synthesis (protein synthesis − protein breakdown) represents milk protein synthesis, with negligible maternal body retention. The true Lys utilization efficiency is the ratio between “Lys in milk” and “Lys for milk”, thus Lys utilized for maintenance should be excluded in the denominator [ 6 ] as follows:

In this study, the whole-body Lys flux was corrected by excluding Lys oxidation (Eq. 16 ), which corresponds to the Lys requirement for maintenance. Guan et al. [ 7 ] reported that Lys flux partitioned to the mammary glands as percentage of whole-body Lys flux was 56% in sows fed a conventional diet, which is comparable to the Lys efficiency values of 50% to 62% observed in this study.

Feeding lactation sows with an improved AA balance diet did not affect milk protein yield and reduced whole-body protein turnover. The reduced whole-body protein turnover resulted from a decrease in both whole-body protein synthesis and breakdown rate, with a tendency for greater protein synthesis to protein breakdown ratio (2.65 vs. 2.02).

Efficiency of Lys was also greater during peak lactation, together suggesting higher efficiency of energy use. These results imply that the lower maternal N retention observed in lactating sows fed improved AA balance diets in previous studies may be a result of greater partitioning of AA towards milk rather than greater body protein breakdown.

Availability of data and materials

All data generated or analyzed during this study are available from the corresponding author on request.

Abbreviations

Average daily gain

Area under the curve

Body weight

Crystalline amino acid

Crude protein

Essential amino acid

Standard error of the mean

Standardized ileal digestibility

3-[methyl- 2 H 3 ]histidine

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This work was financially supported by funds from the USDA-NIFA (award number 2014-67015-21832).

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Department of Animal Science, Michigan State University, East Lansing, 48824, USA

Sai Zhang, Julie Moore & Nathalie L. Trottier

State Key Laboratory of Swine and Poultry Breeding Industry, Key Laboratory of Animal Nutrition and Feed Science in South China, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, PR China

USDA/ARS Children’s Nutrition Research Center and Pediatric Critical Care Medicine, Department of Pediatrics, Baylor College of Medicine, Houston, TX, 77030, USA

Juan C. Marini & Mahmoud A. Mohammad

Department of Large Animal Clinical Sciences, Michigan State University, East Lansing, MI, 48824, USA

Vengai Mavangira & Andrew Claude

Present address: Department of Animal Science, Cornell University, Frank Morrison Hall, 507 Tower Road, Ithaca, NY, 14853-4801, USA

Nathalie L. Trottier

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Conceptualization, NLT and SZ; Methodology, NLT, SZ, JCM, VM, AC, JM, and MAM; Formal analysis, SZ and JCM; Writing, SZ and NLT; Supervision, NLT; Funding acquisition, NLT.

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Correspondence to Nathalie L. Trottier .

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All procedures were approved by the Michigan State University Institutional Animal Care and Use Committee (AUF # 05/16-091-00) and followed the American Association for Laboratory Animal Science guidelines.

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Zhang, S., Marini, J.C., Mavangira, V. et al. Effects of improved amino acid balance diet on lysine mammary utilization, whole body protein turnover and muscle protein breakdown on lactating sows. J Animal Sci Biotechnol 15 , 65 (2024). https://doi.org/10.1186/s40104-024-01020-9

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Received : 07 December 2023

Accepted : 05 March 2024

Published : 07 May 2024

DOI : https://doi.org/10.1186/s40104-024-01020-9

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