John P Whiteman

Seth D Newsome

Paco Bustamante

Yves Cherel

Keith A Hobson

Affiliations

Quantifying capital versus income breeding: New promise with stable isotope measurements of individual amino acids

Keywords: Carbon-13, compound-specific isotope analysis, CSIA, discrimination, fasting, fractionation, nitrogen-15

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Introduction

Allocating stored nutrients to egg formation can alleviate the challenge of accumulating resources at the time or place of reproduction. This 'capital' breeding strategy contrasts with 'income' breeding, in which individuals rely directly on exogenous food sources consumed concomitantly with breeding to build offspring [START_REF] Drent | The prudent parent: Energetic adjustments in avian breeding[END_REF]. Understanding such nutrient allocations during reproduction is valuable because it illuminates the evolution of life-history strategies and the relative importance of different habitats and food sources at different points during the annual cycle, a key factor in organismal responses to environmental shifts [START_REF] Jenouvrier | Projected continent-wide declines of the emperor penguin under climate change[END_REF]. However, tracing allocation of endogenous and exogenous nutrients to reproduction in wild animals is challenging. In birds, these allocations have been assessed using mass balance (reviewed by [START_REF] Hobson | Using stable isotopes to quantitatively track endogenous and exogenous nutrient allocations to eggs of birds that travel to breed[END_REF] and with the measurement of carbon (δ 13 C) and nitrogen (δ 15 N) stable isotope values of diets, endogenous reserves (e.g. skeletal muscle) and egg components [START_REF] Hobson | Using stable isotopes to quantitatively track endogenous and exogenous nutrient allocations to eggs of birds that travel to breed[END_REF][START_REF] Hupp | Spring temperature, migration chronology, and nutrient allocation to eggs in three species of Arctic-nesting geese: implications for resilience to climate warming[END_REF][START_REF] Warner | Lizards flexibly combine stored energy and recently-acquired nutrients to fuel reproduction[END_REF]. The latter approach requires that endogenous reserves not acquired during reproduction at the breeding grounds have a different isotopic composition than the local diet at the time of egg formation (e.g. [START_REF] Hobson | Arctic waders and the capital-income continuum: Further tests using isotopic contrasts of egg components[END_REF]Hobson et al., 2005), and/or that isotopic discrimination associated with the mobilization of endogenous nutrients and egg synthesis yields unique isotopic patterns in egg components [START_REF] Bond | Nutrient allocation for egg production in six Atlantic seabirds[END_REF].

Although stable isotope analysis of bulk tissues has advanced the field, it requires dietary isotopic values from multiple study sites or time points or both. In addition, bulk tissue analysis averages the isotopic composition of all constituent macromolecules. This averaging is problematic because compounds within macromolecules (e.g. amino acids or 'AA' within proteins) participate in different biochemical pathways and can vary widely in isotope values [START_REF] Mcmahon | Amino acid isotope analysis: A frontier in studies of animal migration and foraging ecology[END_REF][START_REF] Whiteman | A guide to using compoundspecific stable isotope analysis to study the fates of molecules in organisms and ecosystems[END_REF]. Animals can synthesize non-essential AAs (AANESS) using carbon from a variety of sources, whereas they cannot synthesize essential AAs (AAESS). As a result, animal tissue AAESS δ13C values reflect primary producers at the base of food webs. Similarly, 'source' AAs retain their amino nitrogen throughout metabolic processing and thus have δ15N values that remain mostly unaltered among primary producers and consumers (O 'Connell, 2017). In contrast, amino nitrogen of 'trophic' AAs can be replaced during metabolic processing; because animals preferentially excrete 14 N in their waste products, their pool of available nitrogen usually has a higher δ 15 N value than dietary sources [START_REF] Balter | Box-modeling of N-15/N-14 in mammals[END_REF], and therefore this replacement causes a systematic increase in trophic AA δ 15 N values with trophic level. Two AAs, Gly and Ser, exhibit δ 15 N patterns that fall between trophic and source AAs (O 'Connell, 2017). Overall, consumer tissue provides information about their diet and trophic ecology via AANESS δ 13 C and trophic AA δ 15 N, and about the base of the food web via AAESS δ 13 C and source AA δ 15 N [START_REF] Whiteman | A guide to using compoundspecific stable isotope analysis to study the fates of molecules in organisms and ecosystems[END_REF].

In this study, we tested AA isotope analysis of body and egg tissues as a novel method to quantify allocation of nutrient sources to eggs in the context of capital versus income reproduction. This method offers the potential benefits of not requiring diet sampling and of having expectations based on well-described anabolic and catabolic pathways for individual AAs. Using samples collected from emperor penguins Aptenodytes forsteri, we compared AA δ 13 C and δ 15 N values among the primary endogenous protein store (skeletal muscle; [START_REF] Robin | Protein and lipid utilization during long-term fasting in emperor penguins[END_REF] and eggs (yolk and albumen). Emperor penguin females fast at their colony for approximately 3 weeks [START_REF] Ancel | The different breeding strategies of penguins: A review[END_REF][START_REF] Groscolas | Metabolic adaptations to fasting in emperor and king penguins, Penguin Biology[END_REF][START_REF] Groscolas | The endocrine control of reproduction and molt in male and female emperor (Aptenodytes forsteri) and Adelie (Pygoscelis adeliae) penguins[END_REF] then synthesize yolk for their single egg over ~21 days, with albumen synthesis on the final day [START_REF] Adams | Embryonic metabolism, energy budgets and cost of production of king Aptenodytes patagonicus and Gentoo Pygoscelis papua penguin eggs[END_REF][START_REF] Ancel | The different breeding strategies of penguins: A review[END_REF]. Yolk protein is primarily derived from liver vitellogenin [START_REF] Deeley | Vitellogenin synthesis in the avian liver[END_REF], and albumen protein from the oviduct [START_REF] Muramatsu | Changes in ovalbumin and protein synthesis in vivo in the magnum of laying hens during the egg formation cycle[END_REF]. Both sites of protein production likely draw their AAs from the body pool of free AAs, which is primarily maintained during fasting by the input of mobilized AAs from skeletal muscle [START_REF] Cherel | Nutrient reserve dynamics and energetics during long-term fasting in the king penguin (Aptenodytes patagonicus)[END_REF][START_REF] Spargo | Metabolic functions of skeletal muscles of man, mammals, birds, and fishes: a review[END_REF]. A capital breeder such as emperor penguins may therefore have similar isotope values for yolk and albumen. In contrast, in income breeding, variation in protein isotope values between yolk and albumen can reflect diet items that were consumed during the different time periods in which these tissues were synthesized [START_REF] Hobson | Arctic waders and the capital-income continuum: Further tests using isotopic contrasts of egg components[END_REF].

We hypothesized that the physiological processes distinguishing capital from income breeding have differential effects on AA δ 13 C and δ 15 N values. Dietary trophic AAs should be subjected to nitrogen discrimination twice in capital breeders. The first instance occurs during assimilation from diet into body tissue, and the second occurs when these AAs are mobilized from body tissue into the endogenous pool of free AAs where they can be used for incorporation into eggs or for biochemical processes such as gluconeogenesis to maintain blood glucose levels during fasting [START_REF] Castellini | The biochemistry of natural fasting at its limits[END_REF][START_REF] Cherel | Physiology and biochemistry of long-term fasting in birds[END_REF][START_REF] Cherel | Relationships between lipid availability and protein utilization during prolonged fasting[END_REF]. This second round of discrimination, during mobilization from endogenous stores (e.g. as fasting penguins 'feed on themselves'; [START_REF] Cherel | Nutrition, physiology, and stable isotopes: New information from fasting and molting penguins[END_REF], should be unique to capital breeders and result in higher δ15N values for trophic AAs in their eggs relative to their endogenous stores than in income breeders. For carbon, income breeders likely source AANESS via the same pathway for both their own tissues and for their eggs, whether routing them intact from the diet or synthesizing them de novo from exogenous carbon.

In contrast, capital breeders should be more likely to synthesize AANESS for their egg tissues using carbon from stored lipids, which provide the largest labile carbon pool in the body and which are 13 C depleted compared to protein and carbohydrates [START_REF] Deniro | Mechanism of carbon isotope fractionation associated with lipid synthesis[END_REF]. In comparison to these expected trends for trophic AAs and AANESS, capital breeding should cause minimal isotopic discrimination for the δ 13 C and δ 15 N values of AAs classified as both essential and source (i.e. Phe and Lys).

The general patterns summarized above lead to specific predictions of how AA isotope values may differ in capital versus income breeding species:

1. In capital breeding, AAESS will be routed from muscle to yolk and albumen without significant isotopic discrimination, resulting in similar δ 13 C values across these tissues. In income breeding, these tissues may or may not have similar δ 13 C values, depending on the δ 13 C values of the dietary sources consumed during egg growth.

2. In capital breeding, AANESS will either be routed from muscle (similar to AAESS) or synthesized de novo for eggs with a combination of protein-and lipid-derived carbon, resulting in similar or lower δ 13 C values in yolk and albumen as compared to muscle. In income breeding, the relationship between δ 13 C values in yolk, albumen and muscle will be unpredictable because AAs may be routed from endogenous tissue or synthesized de novo from a variety of dietary carbon sources.

3. In capital breeding, source AAs will have similar δ 15 N values across muscle and egg tissues if their δ 13 C values suggest direct routing from muscle. In income breeding, source AAs in yolk, albumen and muscle will have δ 15 N values that reflect dietary intake during the period of tissue synthesis.

4. In capital breeding, trophic AAs will have higher δ 15 N values in yolk and albumen than in muscle because of the exchange of their amino nitrogen with the enriched endogenous nitrogen pool. In income breeding, trophic AAs will have similar δ 15 N values across yolk, albumen and muscle because the amino nitrogen in AAs will be subjected to similar metabolic processing in all of these tissues.

As a result of predictions three and four above, we also predict that the offset in δ 15 N values between trophic and source AAs will be higher in egg tissue than in muscle during capital breeding.

The Δ 15 NGlx-Phe offset ('Glx' refers to both glutamine and glutamic acid) in consumer tissue is commonly used as a proxy for trophic level [START_REF] Chikaraishi | Determination of aquatic food-web structure based on compound-specific nitrogen isotopic composition of amino acids[END_REF] because δ 15 NGlx values increase during metabolic processing by consumers at each trophic level, whereas δ 15 NPhe values typically do not (O 'Connell, 2017). Recent studies suggest that Δ 15 NPro-Phe may exhibit a similar trend [START_REF] Mcmahon | Embracing variability in amino acid δ 15 N fractionation: mechanisms, implications, and applications for trophic ecology[END_REF][START_REF] Whiteman | A guide to using compoundspecific stable isotope analysis to study the fates of molecules in organisms and ecosystems[END_REF]. Here, instead of trophic level, we interpret these offsets in yolk and albumen (as compared to offsets in muscle) as proxies for capital breeding that reflect the extent to which muscle AAs provide the raw materials to synthesize AAs in egg tissues. We compare the offsets in emperor penguins to data previously reported for freeranging herring gulls Larus argentatus smithsonianus, an income-breeding species that feeds throughout egg development [START_REF] Hebert | Amino acid specific stable nitrogen isotope values in avian tissues: insights from captive American kestrels and wild herring gulls[END_REF].

Materials and Methods

Field Sampling.

Eggs and skeletal muscle were opportunistically collected in four different years from female penguin carcasses after natural mortalities during egg laying in the breeding colony of Pointe Géologie (66°40′S, 140°01′E), Adélie Land, Antarctica [START_REF] Prévost | R: A language and environment for statistical computing[END_REF]. It was possible in only one instance to collect an egg that was clearly associated with a carcass that was also sampled for skeletal muscle; the data for these paired yolk, albumen and muscle samples are highlighted throughout the results and are presented in combination with data pooled from multiple eggs and muscle samples. Total sample sizes included: yolk and albumen samples from eggs collected in 2011 (N = 3) and 2014 (N = 5); and muscle samples collected in 2008 (N = 3), 2010 (N = 2) and 2011 (N = 3).

Eggs and pectoral muscle from carcasses were stored at -20°C before sample processing. Egg albumen and yolk were separated after thawing the egg. All tissues were freeze-dried then powdered.

Stable Isotope Analysis.

Samples were analysed at the Center for Stable Isotopes (University of New Mexico, Albuquerque, NM, USA). Compound-specific isotopic analysis of individual AAs followed [START_REF] Whiteman | A guide to using compoundspecific stable isotope analysis to study the fates of molecules in organisms and ecosystems[END_REF]. Briefly, samples were weighed out to ~5-20 mg and hydrolysed in 1 ml of 6 N HCl for 20 hr at 110°C. During hydrolysis, asparagine was converted to aspartic acid (Asx) and glutamine was converted to glutamic acid (Glx). The resulting hydrolysate was derivatized to N-trifluoracetic acid isopropyl esters. Samples were injected (1 μl) into a gas chromatograph (Thermo Scientific Trace 1300 GC; column BPx5, 60 m, ID 0.32 mm, film thickness 1.0 μm) and separated AAs were then combusted to CO2 or reduced to N2 at 1,000°C and analysed on an isotope ratio mass spectrometer (Thermo Scientific Delta V Plus).

We injected each sample twice, interspersed with standard injections (AAs of known isotopic values; Sigma-Aldrich Co.) that bracketed either two samples (δ 13 C) or one sample (δ 15 N).

Replicate sample injections had mean SD of 0.2 ‰ for δ 13 C and 0.3 ‰ for δ 15 N values.

Standardization across runs was achieved with intermittent pulses of CO2 or N2 gases of known isotopic value. To account for the addition of carbon to AAs and for the fractionation associated with derivatization, we corrected δ 13 C values as follows:

δ 13 CUSA = (δ 13 CDSA -δ 13 CDST + (δ 13 CUST × P)) × P -1 (equation 1) δ 13 CUSA is the calculated value for the underivatized sample; δ 13 CDSA is the measured value for the derivatized standard; δ 13 CUST is the known value of the underivatized standard; and P is the known proportion of carbon in the AA from the original sample. Correction of δ 15 N values was less complex because derivatization does not add nitrogen atoms to AA:

δ 15 NUSA = (δ 15 NDSA + (δ 15 NDST -δ 15 NUST) (equation 2)
For data analysis (henceforth: yolk and muscle refer to lipid-free samples), we first compared the δ 13 C and δ 15 N values of each AA between yolk and albumen with paired t tests because these tissues were sampled from the same egg. Second, for each AA, we compared δ 13 C and δ 15 N values in muscle among 2008, 2010 and 2011 using a Kruskal-Wallis rank sum test followed by the Dunn's pairwise test. Third, for each AA, we pooled samples from different years and compared δ 13 C and δ 15 N values among muscle, yolk and albumen using a one-way ANOVA followed by a Tukey's HSD.

In this step, we compared the distribution of the pooled data to the data points of the muscle, yolk and albumen that were collected from a single individual. Fourth, we calculated the offsets of Δ 15 NGlx-Phe and Δ 15 NPro-Phe for muscle, yolk and albumen, then compared offsets among tissues with a one-way ANOVA followed by a Tukey's HSD pairwise test. We calculated the same offsets for herring gulls after extracting δ 15 N values for AAs in muscle and egg homogenate (yolk and albumen combined) from figure 3 in [START_REF] Hebert | Amino acid specific stable nitrogen isotope values in avian tissues: insights from captive American kestrels and wild herring gulls[END_REF]. We compared herring Δ15NGlx-Phe and Δ 15 NPro-Phe between muscle and egg homogenate using two-tailed t-tests. We set α = 0.05 and analysed statistics in Program R ( R C ore Team, 2018).

Results

Table 1 summarizes the predicted and observed differences among isotope values for emperor penguin muscle, yolk and albumen.

δ 13 C values Most AA δ 13 C values did not differ between years: across all tissues, 33 of 39 comparisons between years were not significant (Tables S1-S3). In muscle, no AA δ 13 C values differed among 2008, 2010 and 2011; in yolk, δ 13 C differed between 2011 and 2014 for Ser, Lys, Pro and Val; and in albumen, δ 13 C differed between 2011 and 2014 for Phe and Asp. For the AAs that differed between years, three were higher in 2011 and three were higher in 2014. Based on the general consistency of δ 13 C values across years, and that yolk and albumen samples were paired and came from the same egg, we pooled data across years. Using these pooled data, yolk and albumen differed in how their AAESS δ 13 C values compared to muscle (Figure 1). For yolk, four AAESS (Lys, Ile, Leu and Tyr) had δ 13 C values that were similar to muscle, consistent with routing of these AAESS from muscle to yolk. Surprisingly, three AAESS (Thr, Val and Phe) had higher δ 13 C values in yolk than in muscle, indicating that these AAESS either experienced fractionation during their routing from muscle, or were sourced from other endogenous tissue. In contrast to yolk, almost all AAESS (except Thr) in albumen had δ 13 C values that were similar to muscle, suggesting their routing from muscle to albumen.

Yolk and albumen also differed in how their AANESS δ 13 C values compared to muscle (Figure 2).

For yolk, three AANESS (Glx, Asx and Ala) had δ 13 C values that were similar to muscle, suggesting that they were routed between these two tissues. Three AANESS had δ 13 C values that differed between muscle and yolk, indicating that in yolk they had been synthesized with carbon that was either a) lower in δ 13 C value than in muscle and thus potentially lipid-derived (Ser and Gly); or b) higher in δ 13 C value and thus from an unknown source or subjected to fractionation (Pro). For albumen, two AANESS (Pro and Glx) had δ 13 C values that were similar to muscle and therefore appeared to be routed between tissues. The remaining four AANESS (Asx, Ser, Gly and Ala) had lower δ 13 C values in albumen than in muscle, likely reflecting that they had been synthesized with carbon from endogenous lipid stores (e.g. adipose tissue). δ 15 N values Most AA δ 15 N values did not differ between years: across all tissues, 34 of 39 comparisons between years were not significant (Tables S4-S6). In muscle, δ 15 N differed between 2008 and 2010 for Phe; in yolk, δ 15 N differed between 2011 and 2014 for Glu and Thr; and in albumen, δ15N differed between 2011 and 2014 for Lys and Ala. For the AAs that differed between years, one was highest in 2008, two were highest in 2011 and two were highest in 2014. Based on the general consistency of δ 15 N values across years, and that yolk and albumen samples were paired, we pooled data across years, similar to δ 13 C. Using these pooled data, both yolk and albumen had substantially higher δ 15 N values than muscle for Gly and Ser (Figure 3), suggesting that the nitrogen of two AAs came from an endogenous pool with a high δ 15 N value. This origin would be consistent with extensive de novo synthesis of Gly and Ser for yolk and albumen as suggested by their low δ 13 C values relative to that of muscle. Surprisingly, δ 15 N values of the source AAs (Phe, Tyr, Lys) were also higher in yolk and albumen than in muscle, except for Tyr in yolk (Figure 3). Four trophic AAs (Pro, Ala, Val and Leu) had higher δ 15 N values in yolk and albumen than in muscle (Figure 4), suggesting that their amino nitrogen originated from an enriched endogenous pool; Glx exhibited a higher δ 15 N value in yolk than in muscle but not in albumen. For the remaining trophic AAs (Ile and Asx) and Thr, yolk and albumen did not differ from muscle, consistent with routing of these AAs from muscle into the egg. For Glx and Asx, the similarity in their δ 15 N values between muscle and albumen is surprising because their low δ 13 C values relative to muscle suggest that these AAs are synthesized de novo for albumen from 13 C-depleted lipid-derived carbon.

In emperor penguins, Δ 15 NGlx-Phe offsets were similar across muscle, yolk and albumen (one-way ANOVA, p = 0.135, F = 2.205) but Δ 15 NPro-Phe offsets were larger in yolk and albumen than in muscle (one-way ANOVA, p < 0.001, F = 23.300; Figure 5a). In contrast, published data from herring gulls [START_REF] Hebert | Amino acid specific stable nitrogen isotope values in avian tissues: insights from captive American kestrels and wild herring gulls[END_REF] show that both Δ 15 NGlx-Phe and Δ 15 NPro-Phe offsets were similar in muscle and in egg homogenate (Δ 15 NGlx-Phe, t test, p = 0.089; Δ 15 NPro-Phe, t test, p = 0.962; Figure 5b). In other words, for a capital breeder (emperor penguins), Δ 15 NPro-Phe offsets were greater in egg tissue than in muscle, whereas for an income breeder (herring gulls), Δ 15 NPro-Phe offsets were similar in egg tissue and in muscle. This result reflected the general trend for δ 15 N values to be higher in egg tissues than in muscle for trophic AAs (as well as Gly and Ser) in penguins but not in gulls (Figure 6).

Discussion

Overall, our results were consistent with many of our predictions and broadly support the use of differences in AA isotope values between endogenous protein stores and egg tissues to trace nutrient sourcing in the context of capital and income breeding (Table 1). Our data must be interpreted with some caution because samples of emperor penguin eggs and muscle were primarily collected from different individuals in different years. However, results were similar between a) pooled data representing eggs that were not associated with muscle samples from specific penguins, and b) data from egg and muscle samples collected from a single individual. This consistency, in combination with relatively small variation in isotope values across sampling years, suggests that our inferences based on pooled data are robust.

Over half of AAESS had similar δ 13 C values across muscle, yolk and albumen, suggesting that they were routed from endogenous proteins to egg tissues, as expected because emperor penguins cannot synthesize these AAs. Several AAESS differed in δ 13 C values across tissues, which is surprising because these AAs must be obtained from dietary sources; below we discuss the possibility that catabolism-driven fractionation caused this discrimination. In contrast to AAESS, all AANESS except for Glx differed in δ 13 C values between muscle and yolk or between muscle and albumen, as would be expected when these AAs are synthesized de novo to build egg tissue. While source AAs unexpectedly had higher δ 15 N values in yolk and albumen than in muscle, these differences were small in comparison to the differences among tissues in δ 15 N values for trophic AAs, which more readily exchange their nitrogen with the 15 N-enriched body pool.

Our data suggest that overall, the AAs deposited during yolk production experienced more fractionation than the AAs in albumen. Conversely, the AAs in albumen were more likely to have been routed from muscle or synthesized de novo from lipid derived carbon. These inferences indicate that even for a species that is exclusively capital breeding, the sourcing pathways of AAs for protein synthesis in various egg components or fetal tissues can vary. The synthesis of AAs with lipid-derived carbon emphasizes that studies of nutrient allocation during reproduction and fasting should consider carbon transfer between endogenous stores of lipid and protein [START_REF] Mcmahon | Trophic discrimination of nitrogen stable isotopes in amino acids varies with diet quality in a marine fish[END_REF][START_REF] Newsome | Amino acid δ 13 C analysis shows flexibility in the routing of dietary protein and lipids to the tissue of an omnivore[END_REF].

We found that Δ 15 NPro-Phe offsets were substantially larger in egg tissues than in muscle, reflecting the isotopic discrimination that occurs when mobilizing endogenous resources. In comparison, Δ 15 NPro-Phe offsets were similar across egg tissue and muscle in herring gulls [START_REF] Hebert | Amino acid specific stable nitrogen isotope values in avian tissues: insights from captive American kestrels and wild herring gulls[END_REF].

Emperor penguins are representative of capital breeding because their reproductive fast precludes income contribution to egg tissue [START_REF] Ancel | The different breeding strategies of penguins: A review[END_REF]. Although the reproductive strategy of herring gulls is less well-documented, they feed during egg development and are thought to be primarily income breeders [START_REF] Bond | Nutrient allocation for egg production in six Atlantic seabirds[END_REF][START_REF] Hebert | Amino acid specific stable nitrogen isotope values in avian tissues: insights from captive American kestrels and wild herring gulls[END_REF]. Differences in Δ 15 NPro-Phe between gulls and the emperor penguins in this study therefore suggest that nitrogen isotope offsets between this trophic-source pair of AAs provides an index of capital breeding. Further validation of this index is important because use of endogenous stores for reproduction is a fundamental aspect of animal life history, and can constrain how wildlife responds to environmental change [START_REF] Hupp | Spring temperature, migration chronology, and nutrient allocation to eggs in three species of Arctic-nesting geese: implications for resilience to climate warming[END_REF].

Yolk AA origins.

Of the 13 AAs we investigated, seven appeared to be routed from muscle directly into yolk without carbon isotope discrimination. Four of these were likely directly routed because they are AAESS (Ile, Leu, Lys and Tyr), which penguins cannot synthesize de novo. The remaining three were AANESS (Glx, Asx and Ala) that have carbon skeletons which are frequently interconverted with important metabolites (e.g. TCA cycle intermediates). This frequent interconversion could have mixed the carbon in these AAs with larger pools of carbon in other molecules, buffering these AAs from measurable isotope discrimination.

Trophic AAs that were routed directly from muscle to yolk had variable nitrogen dynamics. Ala and Leu appeared to exchange their amino nitrogen with an endogenous pool, resulting in 15 N enrichment of these AAs. This enrichment also occurred for Glx, although the effect size was smaller, perhaps because it was partially buffered by the relatively large pool size of Glx. Asx and Ile did not exhibit 15 N enrichment. Asx may have been buffered from enrichment because frequent transamination caused by its participation in the urea cycle [START_REF] Nelson | Lehninger principles of biochemistry[END_REF] constantly exchanges its amino nitrogen. Ile may have avoided enrichment because its physiological importance as a branched-chain AA can spare it from extensive metabolic processing [START_REF] Holeček | Metabolism of branched-chain amino acids in starved rats: the role of hepatic tissue[END_REF]. It is unclear why the other two branched-chain AA that we measured, Leu and Val, would not have been similarly spared, although it is noteworthy that they share a precursor (alphaketoisovalerate; [START_REF] Nelson | Lehninger principles of biochemistry[END_REF].

Source AAs that were routed directly from muscle into yolk also had variable nitrogen dynamics.

As expected for a source AA, Tyr did not differ between muscle and yolk. However, Phe and Lys were 15 N enriched in yolk. Because emperor penguins had no access to exogenous AAs, we suggest that this enrichment reflected catabolism of Phe and Lys for use as oxidative fuel. Emperor penguins primarily oxidize stored lipids during their breeding fast [START_REF] Robin | Protein and lipid utilization during long-term fasting in emperor penguins[END_REF]; however, some AA catabolism is unavoidable [START_REF] Owen | Protein, fat, and carbohydrate requirements during starvation: anaplerosis and cataplerosis[END_REF]. The complete oxidation of free AAs into CO2 via processes like gluconeogenesis may favour AAs containing the lighter nitrogen isotope. This process would leave AAs with higher δ 15 N values available for deposition into new tissue. It is unclear why this process would not similarly affect the source AA Tyr, although perhaps the ability of animals to synthesize Tyr from Phe [START_REF] Nelson | Lehninger principles of biochemistry[END_REF] reduces enrichment caused by catabolism.

Of the 13 AAs we investigated, six differed in δ 13 C values between muscle and yolk. Of these six, Ser and Gly, both AANESS, had the lowest δ 13 C values and the highest δ 15 N values, suggesting extensive de novo synthesis using lipid-derived carbon and endogenous recycled nitrogen. Widespread de novo synthesis of Gly is plausible because of the high demand for this AA in birds for incorporation into (a) uric acid, their primary nitrogenous waste product [START_REF] Taylor | Dietary glycine: its importance in growth and development of the budgerigar (Melopsittacus undulatus)[END_REF] and (b) eggshell membranes [START_REF] Zhao | Characterization of collagen from eggshell membrane[END_REF]. Because Gly and Ser are readily interconverted via a single reaction, their isotope values tend to be similar (O 'Connell, 2017).

Our results here support the continued distinction of Gly and Ser from other AAs, as their isotope values appear to be sensitive to nitrogen balance and to the primary nitrogen excretionary pathway of the study organism [START_REF] Mcmahon | Embracing variability in amino acid δ 15 N fractionation: mechanisms, implications, and applications for trophic ecology[END_REF][START_REF] Connell | Trophic' and 'source' amino acids in trophic estimation: a likely metabolic explanation[END_REF][START_REF] Whiteman | A guide to using compoundspecific stable isotope analysis to study the fates of molecules in organisms and ecosystems[END_REF].

Of the six AAs that differed in δ 13 C values between muscle and yolk, four had higher δ 13 C values in yolk: the AANESS Pro and the AAESS of Thr, Val and Phe. It is possible that these AAs were used by emperor penguins as fuel for aerobic oxidation resulting in isotopic enrichment as explained above. This explanation would be consistent with the enrichment of both 13 C and 15 N in yolk relative to muscle for Pro, Val and Phe. Intriguingly, higher δ 15 N values of Pro were a sensitive indicator of fasting in southern elephant seals Mirounga leonina [START_REF] Lübcker | Fasting affects amino acid nitrogen isotope values: a new tool for identifying nitrogen balance of free-ranging mammals[END_REF], suggesting that this AA may be particularly responsive to changes in endogenous nitrogen flux. Catabolismdriven discrimination may also explain the enrichment of 13 C in yolk Thr, although the processes governing nitrogen isotopic fractionation for this AA remain unclear [START_REF] Fuller | The dietary protein paradox and threonine 15 N-depletion: Pyridoxal-5'-phosphate enzyme activity as a mechanism for the δ 15 N trophic level effect[END_REF].

Other potential mechanisms to explain higher δ 13 C values in yolk than in muscle for Pro, Thr, Val and Phe seem less likely. First, these AAs could have been routed into yolk from exogenous diet items that were 13 C enriched relative to endogenous muscle. However, in the Southern Ocean primary producer δ 13 C values decline with increasing latitude [START_REF] Lorrain | Nitrogen and carbon isotope values of individual amino acids: a tool to study foraging ecology of penguins in the Southern Ocean[END_REF], therefore the last food items consumed by penguins before their breeding fast were likely relatively low in 13 C.

In addition, penguins fast for 3 weeks before the initiation of yolk formation [START_REF] Ancel | The different breeding strategies of penguins: A review[END_REF], long enough to fully metabolize all food items. Second, AAs could have been mobilized from an endogenous tissue that had a different δ 13 C value than muscle. However, major proteinaceous tissues other than muscle (e.g. liver) tend to have more rapid carbon turnover [START_REF] Bauchinger | Carbon turnover in tissues of a passerine bird: allometry, isotopic clocks, and phenotypic flexibility in organ size[END_REF], which means that the most recently consumed food items should have led to relatively lower δ 13 C values, as explained above. Regardless, skeletal muscle is by far the largest protein reservoir in the body [START_REF] Cherel | Nutrient reserve dynamics and energetics during long-term fasting in the king penguin (Aptenodytes patagonicus)[END_REF][START_REF] Spargo | Metabolic functions of skeletal muscles of man, mammals, birds, and fishes: a review[END_REF]. A third possibility is that AAs were synthesized by gut microbes using alternative carbon sources [START_REF] Freeman | Contributions of direct incorporation from diet and microbial amino acids to protein synthesis in Nile tilapia[END_REF], a phenomenon that appears to occur even in hypercarnivores [START_REF] Whiteman | Amino acid isotope discrimination factors for a carnivore: physiological insights from leopard sharks and their diet[END_REF]. However, this explanation would require routing of new AAs from gut microbes exclusively to yolk, a pathway which we are not aware of.

Albumen AA origins.

In contrast to the 3 weeks required for synthesis of yolk, albumen is produced over a single day [START_REF] Ancel | The different breeding strategies of penguins: A review[END_REF] shortly before egg laying. This rapid production may cause a high demand for routing of AAESS from muscle to albumen, which would explain the similar δ 13 C values between these two tissues for all measured AAESS except Thr. As expected for trophic AAs that were mobilized from muscle, Val and Leu exhibited 15N enrichment in albumen relative to muscle.

The trophic AAs Ile had surprisingly similar δ 15 N values in albumen and muscle, a pattern also observed in the yolk-muscle comparison. As described above for yolk, we speculate that Ile in albumen was spared from metabolic processing because of its role as a branched chain AA [START_REF] Holeček | Metabolism of branched-chain amino acids in starved rats: the role of hepatic tissue[END_REF].

Despite the expectation that source AAs would be routed from muscle with minimal exchange of nitrogen with endogenous pools, Phe, Lys and Tyr had higher δ 15 N values in albumen relative to muscle. We hypothesize that these AAs were enriched in albumen because of fractionation during catabolism for gluconeogenesis, as suggested for yolk. However, unlike yolk, there was no concomitant enrichment in their δ 13 C values, potentially because the time window for catabolism and fractionation is shorter during synthesis of albumen (~1 day) than yolk (~21 days). It is also possible that AA carbon generally experiences less isotopic discrimination than nitrogen because AAs contain several carbon atoms (2-9), whereas most forms only contain a single nitrogen in the amine group. Regardless of the exact mechanisms causing isotopic fractionation, the resulting nitrogen discrimination tended to be smaller for source AAs (0.8-3.1 ‰) than for trophic AAs (0.8-5.3 ‰; see Table 1), which is consistent with previous explanations of the biochemical processes that produce patterns in AA δ 15 N values (O 'Connell, 2017).

The rapid synthesis of albumen may also create a high demand for de novo synthesis of AANESS using the most plentiful endogenous source of carbon: lipids in adipose tissue. Use of 13 C-depleted carbon from lipids would explain why four of six AANESS (Asx, Ser, Gly and Ala) had lower δ 13 C values in albumen than in muscle. For the other two AANESS (Glx and Pro), it is notable that Glx δ 13 C values were lower in albumen than yolk, consistent with at least some synthesis using lipidderived carbon. Because emperor penguins had likely been fasting for nearly 6 weeks at the time of albumen formation [START_REF] Adams | Embryonic metabolism, energy budgets and cost of production of king Aptenodytes patagonicus and Gentoo Pygoscelis papua penguin eggs[END_REF][START_REF] Ancel | The different breeding strategies of penguins: A review[END_REF][START_REF] Groscolas | Metabolic adaptations to fasting in emperor and king penguins, Penguin Biology[END_REF][START_REF] Groscolas | The endocrine control of reproduction and molt in male and female emperor (Aptenodytes forsteri) and Adelie (Pygoscelis adeliae) penguins[END_REF], this de novo synthesis would have used 15 N-enriched, recycled endogenous nitrogen. Accordingly, four AANESS had higher δ 15 N values in albumen than in muscle. The two which did not (Asx and Glx) may have had their δ 15 N values in yolk buffered from substantial change by their relatively large pool sizes in the body, similar to yolk.

Δ 15 N offsets as an index of capital breeding

Almost all trophic AAs exhibited greater offsets between egg tissue and muscle in emperor penguins than in herring gulls, more so than source AAs (see Figure 6). This trend suggests that calculations of Δ 15 NTrophic-Source can be sensitive to the mobilization of endogenous tissue stores.

However, the trophic-source pair of Δ 15 NGlx-Phe, which to date is the most common pairing [START_REF] Mcmahon | Embracing variability in amino acid δ 15 N fractionation: mechanisms, implications, and applications for trophic ecology[END_REF][START_REF] Whiteman | A guide to using compoundspecific stable isotope analysis to study the fates of molecules in organisms and ecosystems[END_REF], did not differ between penguins and gulls.

Instead, Δ 15 NPro-Phe was a better marker: larger values of this offset in egg tissue than in muscle tissue may therefore represent an index of capital breeding. We speculate that Δ 15 NPro-Phe was more sensitive than Δ 15 NGlx-Phe because the relative pool size of endogenous Glx is much larger than that of Pro. As a result, greater mobilization and redeposition should be required of Glx before an isotopic offset of similar magnitude can be achieved. Using Δ 15 NPro-Phe as a quantitative index of reproductive strategy, or any other AA trophic-source pair, requires establishing an expected range of values across a spectrum from complete capital to complete income breeding. We hypothesize that this range of values is broad and contains predictable patterns that reflect animal physiology and ecology, similar to the range of trophic discrimination factors that have been observed when using Δ 15 NGlx-Phe to calculate trophic position [START_REF] Mcmahon | Embracing variability in amino acid δ 15 N fractionation: mechanisms, implications, and applications for trophic ecology[END_REF]. Importantly, fractionation during mobilization of endogenous tissue stores is caused by biochemical

Table 1 .

 1 Predicted and observed differences in δ 13 C and δ 15 N values of individual AAs for egg yolk and egg albumen versus skeletal muscle, in emperor penguins. Each AA is classified based on nitrogen and carbon dynamics; for carbon, NESS is non-essential and ESS is essential. Because tyrosine is synthesized from the essential AA phenylalanine, here it is classified as essential. The 'Predicted' columns indicate the expected direction of the difference between egg tissues and muscle (↔ indicates no difference). The 'Yolk' and 'Albumen' columns indicate the observed direction and magnitude of this difference (three arrows: >6‰, two arrows: 3-6 ‰, one arrow: <3‰); '↔' indicates no significant difference.

Figure 1 .

 1 Figure 1. δ 13 C values for essential AA in skeletal muscle, egg yolk, and egg albumen from emperor penguins. AA are grouped by precursor molecule for de novo synthesis: OXA = oxaloacetate, PYR = pyruvate, PEP = phosphoenolpyruvate. Yolk and albumen samples were from the same egg (N = 8 eggs). Muscle samples (N = 8 penguins) were not paired to yolk and albumen samples with one exception: white circles represent the data for a single penguin from which both muscle and an egg were collected. All samples were collected in 2008, 2010, 2011, and 2014. For rectangles: bars, boxes, error bars, and circles indicate the median, 25 th , 10 th , and 5 th percentiles, respectively.Asterisks indicate significance of comparisons: ***P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05. For each AA, yolk and albumen were compared with a paired t-test, and muscle was compared to yolk and albumen with Tukey HSD pairwise comparisons after a one-way ANOVA.

Figure 2 .

 2 Figure 2. δ 13 C values for non-essential AA in skeletal muscle, egg yolk, and egg albumen from emperor penguins. AA are grouped by precursor molecule for de novo synthesis: TCA = tricarboxylic acid cycle, G3P = glyceraldehyde-3-phosphate, PYR = pyruvate. Yolk and albumen samples were from the same egg (N = 8 eggs). Note that muscle samples (N = 8 penguins) were not paired to yolk and albumen samples with one exception: white circles represent the data for a single penguin from which both muscle and an egg were collected. All samples were collected in 2008, 2010, 2011, and 2014. For rectangles: bars, boxes, error bars, and circles indicate the median, 25 th , 10 th , and 5 th percentiles, respectively. Asterisks indicate significance of comparisons: ***P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05. For each AA, yolk and albumen were compared with a paired t-test, and muscle was compared to yolk and albumen with Tukey HSD pairwise comparisons after a one-way ANOVA.

Figure 3 .

 3 Figure 3. δ 15 N values for AA that are classified as trophic/source (Gly and Ser) or source AA in samples of skeletal muscle, egg yolk, and egg albumen from emperor penguins. Yolk and albumen samples were from the same egg (N = 8 eggs, except N = 3 for Tyr yolk). Muscle samples (N = 8 penguins) were not linked to yolk and albumen samples (i.e., they did not come from the penguin that laid the egg) with one exception: white circles represent the data for a single penguin from which both muscle and an egg were collected. All samples were collected in 2008, 2010, 2011, and 2014. For rectangles: bars, boxes, error bars, and circles indicate the median, 25 th , 10 th , and 5 th percentiles, respectively. Asterisks indicate significance of comparisons: ***P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05. For each AA, yolk and albumen were compared with a paired t-test, and muscle was compared to yolk and albumen with Tukey HSD pairwise comparisons after a one-way ANOVA.

Figure 4 .

 4 Figure 4. δ 15 N values of trophic AA, and separately of Threonine (Thr), in samples of skeletal muscle, egg yolk, and egg albumen from emperor penguins. Yolk and albumen samples were from the same egg (N = 8 eggs). Muscle samples (N = 8 penguins) were not linked to yolk and albumen samples (i.e., they did not come from the penguin that laid the egg) with one exception: white circles represent the data for a single penguin from which both muscle and an egg were collected. All samples were collected in 2008, 2010, 2011, and 2014. For rectangles: bars, boxes, error bars, and circles indicate the median, 25 th , 10 th , and 5 th percentiles, respectively. Asterisks indicate significance of comparisons: ***P < 0.001, **0.001 < P < 0.01, *0.01 < P < 0.05. For each AA, yolk and albumen were compared with a paired t-test, and muscle was compared to yolk and albumen with Tukey HSD pairwise comparisons after a one-way ANOVA.

Figure 5 .

 5 Figure 5. Offsets in  15 N values ( 15 N) between trophic AA (Glx, Pro) and a source AA (Phe). Bars are medians and boxes, error bars, and circles indicate 25 th , 10 th , and 5 th percentiles. (A) Offsets in skeletal muscle, egg yolk, and egg albumen in emperor penguins (N = 8 samples for each tissue type). Asterisks indicate significance of comparisons within Glx-Phe and Pro-Phe groups via Tukey HSD pairwise comparison (*** = P < 0.001) after a one-way ANOVA. (B) Offsets in skeletal muscle and egg homogenate in herring gulls (N = 4); data are from Hebert et al. (2016).

Figure 6 .

 6 Figure 6. Differences in δ 15 N values ( 15 N) between egg tissue and muscle, in an income breeder (herring gull;[START_REF] Hebert | Amino acid specific stable nitrogen isotope values in avian tissues: insights from captive American kestrels and wild herring gulls[END_REF]) and a capital breeder (emperor penguin), for (A) trophic acids (AA) and threonine and for (B) trophic/source (Gly and Ser) and true source AA (Phe, Tyr, Lys). For gulls, egg homogenate and muscle δ 15 N values were compared from the same individual (N = 4). For penguins, the median δ 15 N value was compared between egg yolk or albumen (N = 8) and muscle from unrelated individuals (N = 8), except for one individual which was sampled for both muscle and egg (see text for details). For gulls, bars are medians and boxes, error bars, and circles indicate 25 th , 10 th , and 5 th percentiles. In general, values near 0‰ are expected to be consistent with routing nutrients from the diet to egg tissue, whereas positive values are expected to be consistent with routing endogenous nutrients to egg tissue.

We thank Emma Elliott Smith for input into analytical processes and Alexi Besser and Blanca X. Mora-Alvarez for laboratory assistance. Sample collection was funded by the Institut Polaire Français Paul-Emile Victor (IPEV, programme n°109, H. Weimerskirch) and isotopic analyses were funded by the Center for Stable Isotopes at the University of New Mexico and by a National Science and Engineering Research Council (NSERC) Discovery Grant (2017-04430) to KAH. The IUF (Institut Universitaire de France) is acknowledged for its support to PB as a Senior Member.

Author contributions: JPW, SDN, KAH conceived the ideas and designed methodology; YC and PB provided the samples; JPW conducted isotope analysis and analyzed the data; all authors interpreted the data, contributed critically to the writing, and gave final approval for publication.

All data included in this study are available in Dryad.

mechanisms that are likely consistent across a diversity of species, suggesting that the value of Δ 15 NPro-Phe may be a generalizable predictor of capital breeding.

A potential quantitative index of reproductive strategy is exciting because such an index has proven elusive. While earlier studies of capital breeding provided important advances, they required assumptions about material balance when estimating mass loss during laying (reviewed by [START_REF] Hobson | Using stable isotopes to quantitatively track endogenous and exogenous nutrient allocations to eggs of birds that travel to breed[END_REF] or about variation in dietary isotopic values and isotopic discrimination between endogenous stores and eggs. Our AA approach has the advantage of explicitly quantifying isotopic difference between tissues within a consumer, avoiding these assumptions. Using Δ 15 NPro-Phe values as an index has considerable potential implications for the study of nutrient allocation strategies in breeding birds [START_REF] Stephens | Capital breeding and income breeding: their meaning, measurement, and worth[END_REF], evolution of reproductive strategies [START_REF] Drent | The timing of birds' breeding seasons: the Perrins hypothesis revisited especially for migrants[END_REF], population dynamics in a rapidly changing world [START_REF] Hupp | Spring temperature, migration chronology, and nutrient allocation to eggs in three species of Arctic-nesting geese: implications for resilience to climate warming[END_REF], and the transfer of nutrients and contaminants among biomes by migrating individuals [START_REF] Hebert | Amino acid specific stable nitrogen isotope values in avian tissues: insights from captive American kestrels and wild herring gulls[END_REF]. It is important to note that our inferences were constrained by the limited sample collection that was possible from wild emperor penguins; future studies can provide additional validation by using large sample sizes of eggs and endogenous tissue that are consistently collected from the same individual. We encourage such studies to build on the isotope framework presented here and to evaluate the Δ 15 NPro-Phe offset, as well as other trophic-source pairings of AAs, in avian species spanning the capital versus income spectrum. This spectrum also occurs in non-avian wildlife [START_REF] Stephens | Capital breeding and income breeding: their meaning, measurement, and worth[END_REF], and researchers focused on other taxa should consider applying our approach for their study systems.


PhD, MD Patrice Crochet
email: pcrochet.marseille@gmail.com

MD Antoine Netter

MD Andy Schmitt

MD Anna Garofalo

MD Anderson Loundou

PhD Sophie Knight

MD Benoit Rabischong

PhD, MD Aubert Agostini

Netter

Garofalo Schmitt

Knight

Performance Assessment for Total Laparoscopic Hysterectomy in the Operating Room: Validity Evidence of a Procedure-specific Rating Scale

Keywords: Assessment tool, Laparoscopy, Hysterectomy, Procedural skills, Validity

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

complex procedure that is mainly taught in the operating room (OR) by mentoring [START_REF] Desai | An update on inpatient hysterectomy routes in the United States[END_REF][START_REF] Igwe | Resident participation in laparoscopic hysterectomy: impact of trainee involvement on operative times and surgical outcomes[END_REF]. Yet, there is no consensus on how to assess competence for this procedure. Technical performance can be directly assessed in a structured manner using rating scales. Thus, a qualified observer rates an operator on predefined aspects of operative performance. Analysis of video-recorded procedures using generic global rating scales demonstrated a good correlation between a surgeon's skills and early complications, using criteria such as handling of instruments and respect for tissue [START_REF] Martin | Objective structured assessment of technical skill (OSATS) for surgical residents[END_REF][START_REF] Vassiliou | A global assessment tool for evaluation of intraoperative laparoscopic skills[END_REF][START_REF] Vaidya | Current status of technical skills assessment tools in surgery: a systematic review[END_REF]. However, generic global rating scales are not designed to encompass all the domains of intraoperative performance that lead to safety and long-term effective outcomes. Procedure-specific rating scales were developed to provide informative feedback and to provide assessment criteria that could be linked to the safety and effectiveness of a given procedure [START_REF] Zevin | Development, feasibility, validity, and reliability of a scale for objective assessment of operative performance in laparoscopic gastric bypass surgery[END_REF][START_REF] Bonrath | Comprehensive surgical coaching enhances surgical skill in the operating room: a randomized controlled trial[END_REF][START_REF] Champagne | The American Society of Colon and Rectal Surgeons assessment tool for performance of laparoscopic colectomy[END_REF]. H-OSATS is a detailed procedure-specific rating scale that was designed to assess operative performances of the LH procedure with or without salpingo-oophorectomy, with a view to its potential use for both formative and summative purposes [START_REF] Knight | Development of an objective assessment tool for total laparoscopic hysterectomy: a Delphi method among experts and evaluation on a virtual reality simulator[END_REF].

The objective of this study was to explore the validity evidence of 7 core tasks of the H-OSATS rating scale in the OR setting.

Materials and Methods

This observational cohort study was undertaken at the academic gynecology departments of La Conception Hospital (Marseille) and Arnaud de Villeneuve Hospital (Montpellier) in France. The study was conducted between January 2016 and April 2019. Informed oral consent was obtained from the surgeons assessed. Patients were provided informed consent about the video recordings at the preoperative consultation. All the videos were made anonymous. Ethical approval was obtained from the institutional review board of the French College of Obstetricians and Gynecologists (CEROG 2015-GYN-0801).

Case Selection and Procedure Standardization

LH procedures were recorded in the OR using a standard rod lens laparoscope with a detachable camera. The inclusion criteria were set to ensure a homogeneous level of difficulty among the operators: simple cases of patients presenting with a pear-shaped uterus (maximum size: resembling 8 weeks' pregnancy) involving minimal or no pelvic adhesion. All cases were undertaken using a uterine manipulator: Clermont-Ferrand (Karl Storz, Tuttlingen, Germany) or RUMI II (CooperSurgical, Trumbull, CT). The type of energy device used was left to the discretion of the surgeon (bipolar electrosurgery or ultrasonic). The operator was always placed on the left side of the patient.

List of Tasks Assessed

Six core tasks of the LH procedure were considered, as defined by the procedure-specific H-OSATS rating scale [START_REF] Knight | Development of an objective assessment tool for total laparoscopic hysterectomy: a Delphi method among experts and evaluation on a virtual reality simulator[END_REF]: (1) division of the round ligaments, (2) creation of the bladder flap, (3) opening of the posterior peritoneum, (4) division of the uterine vessels, (5) colpotomy, and (6) retrieval of the uterus and closure of the vault. A seventh task performed in the case of LH with salpingo-oophorectomy was evaluated: division of the infundibulopelvic (IP) ligament. Three tasks were unilaterally considered (rightor left-sided): (1) division of the round ligament, (2) division of the IP ligament, and (3) opening of the posterior peritoneum. All "division of the uterine vessels" videos were left-sided because we assumed that the evaluation of this task was easier on the left side in the context of a blinded video review. Only 2 out of the 5 items included in the "retrieval of the uterus and closure of the vault" videos were retained for skills assessment on the videos, and consequently this task was relabeled "closure of the vault." To allow a more accurate comparison among the operators, all "closure of the vault" videos concerned cases where the uterosacral ligaments' insertion into the vagina was intact after colpotomy.

Participants

Three groups of surgeons with different levels of experience (novice, intermediate, and experienced surgeons) were video recorded during LH performances. The inclusion criteria for the novice group were defined as follows: postgraduate gynecology residents (from year 3 to 5), attendance at a lecture on a step-by-step description of the LH procedure, open access to a didactic training presentation and a video demonstration of an experienced surgeon performing the LH procedure, prior completion of the fundamentals of laparoscopic surgery curriculum on a fundamentals of laparoscopic surgery box trainer during a 6month rotation in a gynecology department, and prior experience as a primary operator in 1 to 4 LH procedures [START_REF] Sroka | Fundamentals of laparoscopic surgery simulator training to proficiency improves laparoscopic performance in the operating room-a randomized controlled trial[END_REF]. Videos from the intermediate group were obtained from first-and second-year attending surgeons who had performed between 10 and 50 LH procedures independently. Videos from the experienced group were obtained from attending surgeons with more than 5 years of experience in an academic hospital and roles in resident and young attending surgeons' education. They had performed more than 200 LH procedures.

Video Selection

Videos from the novice group were selected using the following criteria: the task was entirely performed by a novice surgeon without any proactive guidance from the attending surgeon. When guidance of the attending surgeon was necessary to ensure patient safety, the degree of this intervention was evaluated by the attending surgeon at the end of the procedure using the Zwisch scale. This scale defines the level of guidance provided to the resident during a case, and it has 4 levels: [START_REF] Feldman | SAGES video-based assessment (VBA) program: a vision for life-long learning for surgeons[END_REF] show and tell, (2) active help, (3) passive help, and (4) supervision only [START_REF] George | Reliability, validity, and feasibility of the Zwisch scale for the assessment of intraoperative performance[END_REF]. All videos requesting more than passive help were excluded. Consequently, videos from the novice group selected for evaluation could be extracted using video procedures where the operator had performed only certain chosen tasks. Surgeons from the intermediate and experienced groups received no guidance during the procedures.

Eligible videos of the LH procedures were prospectively collected. One of the authors (P.C.) screened each video to determine which tasks were appropriate for video review (i. e., tasks recorded from the beginning to the end with no cut). These videos were subsequently edited to obtain the full sequence of eligible tasks. Data collection ended when 9 to 11 videos of each task were obtained for each of the 3 groups with different levels of experience. Among the eligible videos of tasks, there was a video of an organ injury that occurred (e.g., bladder) during a procedure performed by an operator from the intermediate group. This video was excluded from the analysis because of a potential bias in favor of the novice group that operated under guidance for safety reasons.

Assessment

All the videos were assessed independently by 2 raters using the H-OSATS rating scale. These raters were attending surgeons at a teaching hospital. They had completed more than 50 LHs and had experience in teaching LH at their institution to trainees as well as in society-related courses. Each item of a specific task was rated from 1 to 5 (with 1 indicating an item not performed or dangerously performed, 3 indicating an item performed adequately with no obvious danger for the patient, and 5 indicating an ideal performance). The raters assessed the videos and applied these rating criteria to each item independently of each other. A calibration session was held before the video-based assessment to ensure that the interpretation of performances as defined by the scale was similar for the 2 raters. Any identifiers were removed from the laparoscopic videos, and the raters were blinded to the identity of the operator. The order of the videos to be reviewed was changed for each round of ratings.

Validity Evidence

Validity evidence for the evaluation of each of the 7 tasks was assessed in accordance with Messick's conceptual framework of validity [START_REF] Association | NCME. Standards for Educational and Psychological Testing[END_REF][START_REF] Ghaderi | Technical skills assessment toolbox: a review using the unitary framework of validity[END_REF]. Evidence of content had been established by agreement among a Delphi panel of 14 international experts, which was published by our team in 2018 [START_REF] Knight | Development of an objective assessment tool for total laparoscopic hysterectomy: a Delphi method among experts and evaluation on a virtual reality simulator[END_REF]. The response process was ensured by the calibration session between the 2 raters. We examined the validity evidence of 3 other main sources of validity: (1) relationship to other variables, (2) internal structure, and (3) consequences. Evidence to support the relationship to other variables was explored by comparing the scores of the 3 groups having different levels of experience. The proof of internal structure was provided by evaluating the reliability of the H-OSATS rating scale: the inter-rater reliability was assessed by correlating scores between the 2 independent raters. The test-retest reliability was evaluated by correlating the scores assessed at 2 different time points-at least 1 month apart-by 1 of the raters. The raters were asked not to spend more than 6 hours per day performing the video reviews to avoid evaluation discrepancies due to boredom. Evidence of consequences was based on a pass/fail competency standard, which was established using the contrasting group method [START_REF] Jørgensen | Contrasting groups' standard setting for consequences analysis in validity studies: reporting considerations[END_REF][START_REF] Szasz | Setting performance standards for technical and nontechnical competence in general surgery[END_REF]. The performance of the participants was first scored by the rater using the H-OSATS rating scale, as explained previously. One month later, the same rater assessed the overall performance of the task, providing a competent/noncompetent classification. In the context of the present study, competence was defined as the ability to safely and effectively perform a task. In case of a disagreement between the 2 raters, a third rater assessed the performance to assign it to the competent/noncompetent group. Next, the operators were divided into 2 contrasting groups (i.e., competent and noncompetent). When using the contrasting group method, the cutoff point was set by identifying the intercept of the normally distributed curves that represented the task-score distributions of the groups defined by their competent/noncompetent status. Theoretical false-positive and false-negative figures were reported because of the small group sizes. Indeed, observed falsepositive and false-negative rates are very sensitive to small samples and outliers [START_REF] Jørgensen | Contrasting groups' standard setting for consequences analysis in validity studies: reporting considerations[END_REF].

Statistics

The results were reported as their mean values and standard derivation. The sample size was calculated using an analysis of variance in PASS 2008 software v.08.0.7 (NCSS, LLC, Kaysville, UT), with the hypothesis of a mean OSATS score of 25 (out of 30) in the experienced group, 20 in the intermediate group, and 13 in the novice group (80% statistical power, type I error rate of 5%). The standard deviation of mean values was assumed to be 4.92, and the common standard deviation within a group was assumed to be 8.00. These values were chosen on the basis of the results of a prior study on LH [START_REF] Crochet | Development of an evidence-based training program for laparoscopic hysterectomy on a virtual reality simulator[END_REF]. Using these criteria, 10 participants had to be recruited in each group. To compare scores among the groups and control for the variation coming from the centers, a linear mixed model was used. The inter-rater reliability was assessed by correlating the total and item scores between the 2 independent raters, using the intraclass correlation coefficient (ICC) (2way mixed-effects model, absolute agreement). The test -retest reliability was assessed by correlating the scores assessed at 2 different time points by the same rater, using the ICC. Data were analyzed using SPSS v.20.0 (IBM Corp., Armonk, New York), and p <.05 was considered statistically significant.

Results

Operative Time

For each task, the mean operative times of the video sequences and comparison of the 3 groups with different levels of experience are presented in Table 1.

Relation to Other Variables

The task scores and comparison of the 3 groups are provided in Table 2. The total scores were different for all tasks, except "division of the IP ligament." Each individual item score was significantly different for the tasks "creation of the bladder flap," "opening of the posterior peritoneum," "colpotomy," and "closure of the vault." None of the items regarding lateral uterine manipulation found a difference among the 3 groups, except for "creation of the bladder flap."

A group-by-group comparison is provided in Supplemental Table 1 for all tasks. The total scores were higher in the experienced group than in the intermediate group for "creation of the bladder flap," "opening of the posterior peritoneum," "colpotomy," and "closure of the vault." All item scores were higher in the experienced group than in the intermediate group for "opening of the posterior peritoneum," "colpotomy," and "closure of the vault." None of the item scores differed between the experienced and intermediate groups for "division of the IP ligament." The total scores were higher in the intermediate group than in the novice group for "creation of the bladder flap," "division of the uterine vessels," and "closure of the vault." All item scores were higher in the intermediate group than in the novice group for "closure of the vault." None of the item scores for "division of the IP ligament," "opening of the posterior peritoneum," and "colpotomy" were different in the intermediate group compared with the novice group.

Sixty-four percent (139/217) of the videos were recorded at the first center (Marseille) and 36% (78/217) at the second center (Montpellier). The estimation of variance due to the center effect was 19% for the "division of the round ligament" task and 0.1% for the "closure of the vault" task, and there was no variance for the 5 other tasks.

Internal Structure

The inter-rater reliability ICCs for total scores were >0.7 for all tasks, except "opening of the posterior peritoneum" (Table 3). These coefficients for individual items of a specific task ranged from 0.389 (p = .090) to 0.861 (p ≤.001). Five out of the 7 items related to uterine manipulation or exposure had an inter-rater reliability ICC of <0.6. The test -retest reliability coefficients for total scores were >0.7 for all tasks, except "opening of the posterior peritoneum." These coefficients for individual items of a specific task ranged from 0.437 (p = .049) to 0.962 (p <.001).

Consequences

The cutoff scores obtained by the contrasting group method for each of the 7 tasks are displayed in Fig. 1. Theoretical false-positive and false-negative rates are presented in Table 4. "Creation of the bladder flap," "colpotomy," and "closure of the vault" had a theoretical false-positive rate of <10%. "Colpotomy" had a theoretical false-negative rate of <10%. "Division of the IP ligament" and "opening of the posterior peritoneum" had a theoretical false-positive or false-negative rate of >25%. Details about competence status (sorted by groups with different levels of experience) and observed false-negative and false-positive rates can be found in Supplement Table 2.

Discussion

This study gathered the validity evidence of an assessment tool specific to LH with salpingo-oophorectomy in the .118 Optimize exposure of the uterine vessels (out of 5)

3.9 (0.6) 3.9 (0.9) 4.3 (0.4) .199 Skeletonize uterine vessels at the ascending portion of the uterine artery (out of 5) 2.5 (0.8) 3.4 (0.9) 3.8 (0.8) <.001 Coagulate the uterine vessels using an appropriate energy source or suture (out of 5)

2.8 (0.9) 3.6 (1.1) 3.6 (0.9) .038 Section uterine vessels in the ascending portion, at the level of the colpotomizer (out of 5)

2.8 (0. OR. Separate evaluations of the core tasks of the procedure allowed exploration of the assessment potential of each task independently, with minimal interference from what was performed before. Validity evidence differed across the tasks. Three tasks (i.e., creation of the bladder flap, colpotomy, and closure of the vault) emerged by their good results across the 3 tested aspects of validity: (1) a meaningful total score difference among the 3 groups with different levels of experience and between the 2 groups of attending surgeons, (2) reliability outcomes of >0.7, and (3) a pass/fail score obtained with a theoretical false-positive rate of <10%. These 3 tasks have common characteristics that may explain these outcomes: first, the operative time was higher than for the other tasks, which gives the rater more comfort in making a judgment. Second, a good understanding of anatomic landmarks is crucial for these tasks, and any error or approximation is easily spotted by the rater. Finally, these tasks require a high level of generic skills. Surprisingly, "division of the IP ligament" could not be discriminated among the 3 groups with different levels of experience. This can be explained not only because different techniques were allowed (with or without fenestration), but also because it was not easy to ascertain from a mute video-based assessment whether the surrounding structures were identified by the operator. The "division of the uteroovarian ligament" task, performed in cases of hysterectomy without salpingo-oophorectomy, was not considered in this study and needs further investigation. The overall compelling findings of reliability, using only 2 raters, showed that the tool is suitable for providing formative feedback because a reliability coefficient of >0.70 is deemed acceptable [START_REF] Downing | Reliability: on the reproducibility of assessment data[END_REF]. The inter-rater reliability may be improved by increasing the number of raters or rated procedures per surgeon [START_REF] Konge | Using virtual-reality simulation to assess performance in endobronchial ultrasound[END_REF].

Tremblay et al [START_REF] Tremblay | Assessment tool for total laparoscopic hysterectomy: a Delphi consensus survey among international experts[END_REF] published a comprehensive rating scale specific to the LH procedure, but no validity evidence was provided in the surgical setting. Recently, Savran et al [START_REF] Savran | Objective assessment of total laparoscopic hysterectomy: development and validation of a feasible rating scale for formative and summative feedback[END_REF] published a short version of a procedure-specific scale. This scale revealed sound validity evidence when used for a full procedure in the OR with 2 groups of operators: beginners and experienced surgeons. Application of this scale for summative purposes needs further evidence because the beginners scored quite low, and it was unclear which level of guidance was received by this group.

An ideal evaluation of technical skills for LH should include all tasks of a full-length procedure. In this case, proficiency would be achieved only if all tasks were individually rated above the cutoff scores. However, certain tasks (i. e., creation of the bladder flap, colpotomy, and closure of the vault) demonstrated higher validity evidence than others (e.g., division of the IP ligament). A peer review limited to these 3 specific tasks could be advantageous to focus the rater's attention on the more discriminative part of the procedure. This hypothesis should be confirmed by further studies. In addition, the postoperative consequences of the ratings should be further investigated because the cutoff scores were based on an intraoperative evaluation of competence. Although performances below the cutoff score can potentially be predictive of increased adverse events or costs, this study did not provide evidence on this matter. 3.0 (0.6) 3.0 (0.9) 4.4 (0.6) <.001

Check that there are no interposed elements around the vaginal fornices, and complete dissection if necessary (out of 5)

2.8 (0.8) 3.0 (0.9) 4.3 (0.6) <.001

Proceed to circumferential colpotomy using an appropriate energy source (out of 5)

2.5 (0.5) 3.0 (0.9) 4. The H-OSATS scale was revealed to be a valuable formative assessment tool in the OR for simple cases. In their practice, surgeons are often confronted by a wider spectrum of anatomic presentations that were not tested in the present study. Consequently, this scale cannot be recommended as the sole certification tool for the LH procedure. Its role in a future multimodal process of summative assessment needs to be further investigated.

The strength of the present study lies in the incremental levels of experience of the 3 groups of operators. The participation of the novice group with minimal guidance was ethically possible only because the scale was evaluated task by task. Careful selection of video-recorded task performances in the novice group could not fully eliminate the guidance bias that remained for safety reasons. Another limitation was the absence of information regarding the handedness of the operators, given that all operators stood on the left side of the patient. Assessment through a video interface limits the scope of evaluation of operative skills [START_REF] Madani | What are the principles that guide behaviors in the operating room?: creating a framework to define and measure performance[END_REF]. Thus, some facets of technical competence, such as setting exposure, can be difficult to measure by watching an internal video feed alone [START_REF] Champagne | The American Society of Colon and Rectal Surgeons assessment tool for performance of laparoscopic colectomy[END_REF]. This was confirmed in this study by the poor performance of items related to exposure or uterine manipulation. Finally, the broad range of experience permitted in the intermediate group (10 to 50 LHs) implied that some participants had not completed their learning curve. Thus, some participants did not reach the cutoff score for competence in this group. However, the scale found differences in performance between the 2 groups of attending surgeons (i.e., <2 years or >5 years of experience) for most of the tasks.

Conclusion

The H-OSATS rating scale proved to be a promising video-based assessment tool in the OR for 3 core parts of the procedure: (1) creation of the bladder flap, (2) colpotomy, and (3) closure of the vault. Other parts of the H-OSATS rating scale showed mixed outcomes and could probably not be considered individually for high-stakes assessment. The next steps would involve confirming the value of H-OSATS individual tasks in a multicenter setting and exploring the assessment potential of this scale during full-length procedures in the OR.

Table 4

Theoretical false-positive and false-negative rates for the 7 core tasks of the laparoscopic hysterectomy procedure, calculated using the normally distributed curves that represent the score distributions of the competent and noncompetent groups False-positive rate, % False-negative rate, %

  Identify the cervicovaginal delineation from the colpotomizer (out of 5)

Fig. 1

 1 Fig. 1Contrasting group curves for each of the 7 core tasks of laparoscopic hysterectomy with salpingo-oophorectomy. The pass/fail scores are represented by the dashed line.

Table 1

 1 Operative time (in minutes) of the 7 core tasks of the laparoscopic hysterectomy procedure. Results are presented as the mean (standard derivation).

	Comparison of the 3 groups of operators by a linear mixed model
	H-OSATS tasks	Novice	Intermediate	Experienced	p-value
	Division of the round ligament	4.8 (2.4)	2.4 (1.2)	1.7 (0.9)	<.001
	Division of IP ligament	8.6 (8.4)	5.1 (2.7)	3.7 (2.2)	.112
	Creation of the bladder flap	19.6 (7.1)	10.9 (5.7)	6.7 (4.6)	<.001
	Opening of the posterior peritoneum	3.2 (2.2)	2.7 (1.2)	2 (1)	.210
	Division of the left uterine vessels	14.3 (9)	6.6 (3.6)	5 (1.9)	.001
	Colpotomy	21.5 (7.8)	17.2 (7.4)	9 (4.2)	.001
	Closure of the vault	32.5 (8)	22.5 (7)	15.4 (5.3)	<.001

Table 2

 2 Scores obtained by the 3 groups of operators for the 7 tasks of laparoscopic hysterectomy with salpingo-oophorectomy and corresponding items. Results are presented as mean (standard derivation). Comparison by a linear mixed model

	Division of the round ligament
		Novice	Intermediate	Experienced	p-value
		N = 10	N = 9	N = 11
	Manipulator: push uterus cranially and laterally toward the opposite side (out of 5)	3.7 (1.3)	3.3 (1.1)	3.7 (1.0)	.861
	Coagulation and transection of the round ligament (out of 5)	3.5 (1.1)	4.1 (0.5)	4.6 (0.5)	<.001
	Individualize the anterior and posterior leaf	3.3 (0.6)	3.8 (0.6)	4.1 (0.5)	<.001
	of the broad ligament (out of 5)
	Total (out of 15)	10.4 (2.7)	11.2 (1.9)	12.4 (1.8)	.022
	Division of the IP ligament
		Novice	Intermediate	Experienced	p-value
		N = 10	N = 11	N = 10
	Manipulator: push uterus cranially and laterally	2.9 (1.1)	2.8 (0.9)	3.3 (0.9)	.423
	toward the opposite side (out of 5)
	Expose IP ligament (out of 5)	3.5 (1.0)	3.6 (0.6)	3.9 (0.9)	.531
	Ureter identification (out of 5)*	2.7 (1.4)	3.4 (1.4)	3.8 (1.1)	.042
	Coagulate using an appropriate energy source or suture (out of 5)	3.5 (1.0)	3.7 (0.9)	3.8 (1.0)	.708
	Section the IP ligament (out of 5)	3.5 (0.9)	3.6 (0.6)	4.1 (0.9)	.082
	Total (out of 25)	16.0 (4.5)	17.1 (3.0)	18.6 (3.7)	.155
	Creation of the bladder flap
		Novice	Intermediate	Experienced	p-value
		N = 11	N = 9	N = 10
	Manipulator: push uterus cranially and laterally to the opposite side	6.4 (1.4)	6.6 (1.4)	7.7 (1.3)	.046
	(left and right) (out of 10)
	Open the anterior leaf of the broad ligament on both sides	5.3 (1.4)	7.4 (1.0)	7.9 (1.3)	<.001
	down to the level of the vesicouterine reflection (out of 10)
	Manipulator: push uterus cranially and posteriorly (out of 5)	2.8 (0.7)	3.4 (0.7)	4.3 (0.5)	<.001
	Section the peritoneum in front of the lower uterine segment (out of 5)	2.6 (0.6)	3.6 (0.9)	4.2 (0.6)	<.001
	The bladder is grasped at the midline, applying an anterior-superior traction (out of 5)	2.5 (0.5)	3.2 (0.5)	4.2 (0.5)	<.001
	Opening of the vesicouterine space at the midline until the	2.5 (0.7)	3.6 (0.9)	4.2 (0.6)	<.001
	cervicovaginal margin is exposed (out of 5)
	Total (out of 40)	22.4 (3.9)	27.7 (4.2)	32.3 (3.4)	<.001
	Opening of the posterior peritoneum

  -OSATS = objective scale specific for the assessment of technical skills for laparoscopic hysterectomy; IP = infundibulopelvic. * With or without fenestration as described in the H-OSATS scale.

				1 (0.6)	<.001
	Total (out of 20)	11.9 (1.9)	12.4 (3.2)	17.1 (1.5)	<.001
	Closure of the vault
		Novice	Intermediate	Experienced	p-value
		N = 11	N = 10	N = 11
	Suture the vaginal vault with interrupted or continuous	1.9 (0.4)	2.8 (0.8)	4.1 (0.5)	<.001
	sutures (out of 5)
	Vaginal suture including sufficient width of vaginal mucosa	2.0 (0.5)	2.8 (0.7)	4.0 (0.7)	<.001
	and fascia (out of 5)
	Total (out of 10)	3.9 (0.7)	5.6 (1.2)	8.1 (1.0)	<.001

H

Table 3

 3 Reliability coefficients for total scores of the 7 core tasks of laparoscopic hysterectomy with salpingo-oophorectomy

	Inter-rater	Test-retest

Table 1

 1 Group by group comparison of performances (mean + standard derivation) between novice, intermediate and experienced groups (Comparison by a linear mixed model)

	Division of the round ligament	14.3	13.6
	Division of the IP ligament	28.4	15.9
	Creation of the bladder flap	4.8	9.7
	Opening of the posterior peritoneum	15.8	25.5
	Division of the uterine vessels	16.9	15.1
	Colpotomy	8.7	16
	Closure of the vault	3.8	10.3

Journal of Minimally Invasive Gynecology. Vol 28, No 10, October 2021

The authors declare that they have no conflict of This study was funded by the Assistance Publique-Hôpitaux de Marseille. This study was approved by the institutional review board of the French College of Obstetricians and Gynecologists (CEROG 2015-GYN-0801; August 31, 2015). The authors confirm that the data supporting the findings of this study are available within the article and its appendices.

Supplemental Table 1