Stéphane Culine
email: stephane.culine@aphp.fr

Valentin Harter

Gwenaelle Gravis

Aude Fléchon

Christine Chevreau

Hakim Mahammedi

Brigitte Laguerre

Aline Guillot

Florence Joly

Sophie Abadie-Lacourtoisie

Lionnel Geoffrois

Frédéric Di Fiore

Guilhem Roubaud

Philippe Barthélémy

Eric Voog

Sheik Emambux

Camille Serrate

Carolina Saldana

Thierry Nguyen-Tan-Hon

Yohann Loriot

Jean-Christophe Eymard

Olivier Huillard

Frédéric Rolland

Nadine Houédé

Jean-Philippe Spano

Mounira El Demery

Sabine Vieillot

Tifenn L'haridon

Werner Hilgers

Yves Allory

Christian Pfister

Vesper Trial

Chemotherapy for muscle-invasive bladder cancer: impact of cisplatin delivery on renal function and local control rate in the randomized phase III VESPER (GETUG-AFU V05) trial

Keywords: Muscle-invasive bladder cancer, cisplatin-based chemotherapy, neoadjuvant treatment, pathological complete response

A deep analysis of data from a randomized trial of perioperative chemotherapy in muscleinvasive bladder cancer shows that a minimum number of 4 cycles is required to optimize the chances of pathological complete response at cystectomy. Increasing the number beyond 4 cycles does not lead to a clinically significant deterioration in renal function without obvious gain on pathological complete response.

Background: Cisplatin-based combination chemotherapy before surgery is the standard of care for muscle-invasive bladder cancer. However, the optimal chemotherapy modalities have not been precisely defined to date.

Conclusion:

At least 4 cycles of cisplatin-based chemotherapy should be delivered before cystectomy. Increasing the number of cycles beyond 4 cycles does not lead to a clinically significant deterioration in renal function but without obvious gain on local control.

Patients and Methods: In the VESPER trial, patients received after randomization either gemcitabine and cisplatin (GC, 4 cycles) or methotrexate, vinblastine, doxorubicin and cisplatin (dose dense [dd]-MVAC, 6 cycles). Creatinine clearance (CrCl) was calculated before each cycle according to the Cockroft and Gault formula. Definition criteria for local control after neoadjuvant chemotherapy included pathological complete response (ypT0N0), pathological downstaging (<ypT2N0) and organ-confined disease (<ypT3N0) at cystectomy.

Baseline and treatment characteristics were studied in multivariate logistic models to determine their potential role for each type of pathological responses.

Results: A total of 2,128 cycles of chemotherapy were delivered, including 2,120 (99.6%) with cisplatin. Full doses of cisplatin were given in 1866 (88%) cycles. Twenty-three (4.7%) patients had to stop chemotherapy (12 GC, 11 dd-MVAC) because of renal failure. No difference in CrCl median values was observed between the two regimens during the first four cycles. A mild decrease occurred thereafter in patients treated with two additional cycles of dd-MVAC. A minimum total dose of 270 mg/m 2 for cisplatin was mandatory to optimize pathological complete responses.

Introduction

Primary surgical management of muscle-invasive bladder cancer (MIBC) with radical cystectomy and extended pelvic lymph node dissection can usually cure 50% of patients.

Neoadjuvant cisplatin-based combination chemotherapy (NAC) before surgery has demonstrated a survival benefit based on level 1 evidence, with regimens including cisplatin, methotrexate, vinblastine with (MVAC) or without (CMV) doxorubicin. [START_REF] Grossman | Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer[END_REF][START_REF]Advanced Bladder Cancer Meta-analysis Collaboration. Neoadjuvant chemotherapy in invasive bladder cancer: update of a systematic review and meta-analysis of individual patient data[END_REF][START_REF]International collaboration of trialists. International phase III trial assessing neoadjuvant cisplatin, methotrexate, and vinblastine chemotherapy for muscleinvasive bladder cancer: long-term results of the BA06 30894 trial[END_REF] However due to significant toxicities, alternative combinations such as dose dense MVAC (dd-MVAC) and gemcitabine-cisplatin (GC) have been proposed over the last two decades. Although different retrospective studies suggested better outcomes with dd-MVAC, [START_REF] Zargar | Neoadjuvant dose dense MVAC versus gemcitabine and cisplatin in patients with cT3-4aN0M0 bladder cancer treated with radical cystectomy[END_REF][START_REF] Peyton | Downstaging and survival outcomes associated with neoadjuvant chemotherapy regimens among patients treated with cystectomy for muscle-invasive bladder cancer[END_REF] no prospective randomized trial comparing the respective impact on survival of dd-MVAC and GC had been reported so far. In the phase III VESPER trial recently conducted by The French Genitourinary Tumor Group (GETUG), patients received either regimen after randomization. More severe asthenia, gastrointestinal side effects and anemia along with a higher control rate were observed in the dd-MVAC arm. [START_REF] Pfister | Randomized phase III of dose dense methotrexate, vinblastine, doxorubicin and cisplatin (dd-MVAC) or gemcitabine and cisplatin (GC) as peri-operative chemotherapy for patients with muscle-invasive bladder cancer (MIBC). Analysis of the GETUG/AFU V05 VESPER trial secondary end-points: chemotherapy toxicity and pathological responses[END_REF] In the present report, the authors focused on renal function during chemotherapy and investigated the role of cisplatin delivery (total dose, dose intensity) on local control rate.

Patients and Methods

Patients and chemotherapy protocol

As previously described, 500 MIBC patients were included in the VESPER trial from February 2013 to March 2018 in 28 French participating centres after informed signed consent (Clinical trial registry: clinicaltrials.gov -NCT 018 12369). [START_REF] Pfister | Randomized phase III of dose dense methotrexate, vinblastine, doxorubicin and cisplatin (dd-MVAC) or gemcitabine and cisplatin (GC) as peri-operative chemotherapy for patients with muscle-invasive bladder cancer (MIBC). Analysis of the GETUG/AFU V05 VESPER trial secondary end-points: chemotherapy toxicity and pathological responses[END_REF] Key inclusion criteria were histologically confirmed muscle-invasive urothelial carcinoma and disease defined by a cT2, cT3 or cT4a N0 (lymph node ≤ 10 mm on CT scan) M0 staging for patients receiving NAC or pT3 or pT4 or pN+ regardless of pT stage and M0 for patients receiving adjuvant chemotherapy.

Patients received after randomization either gemcitabine 1250 mg/m 2 on day 1 and day 8 and cisplatin 70 mg/m 2 on day 1 every 3 weeks for a total of 4 cycles (GC regimen) or methotrexate 30 mg/m 2 on day 1, vinblastine 3 mg/m 2 on day 2, doxorubicin 30 mg/m 2 on day 2 and cisplatin 70 mg/m 2 on day 2 with G-CSF from day 3 to day 9, every 2 weeks for a total of 6 cycles (dd-MVAC regimen). In both arms, creatinine clearance (CrCl) was calculated before each cycle according to the Cockroft and Gault (CG) formula: CrCl = (140-age) x (weight in kg) x (0.85 if female) / (72 x serum creatinine). A CrCl of 50 mL/min or more was required to enter the trial and start chemotherapy with full standard doses. The following adaptations were done in subsequent cycles for cisplatin according to CrCl: ≥ 60 mL/min, 70 mg/m 2 ; between 50 and 59 mL/min, 50 mg/m 2 ; between 40 and 49 mL/min: 40 mg/m 2 ; < 40 mL/min: end of treatment.

Regarding neutropenic fever or other grade ≥ 3 toxicities, a 15% dose reduction was done for all drugs in both arms. In the absence of toxicity recovery within 14 days, or in case of grade 4 toxicity despite a dose reduction of 15%, chemotherapy was stopped.

Renal impairment during chemotherapy was defined by the threshold of a ClCr requiring cisplatin dose reduction, i.e. < 60 mL/min. The following criteria were used for efficacy on bladder cancer: pathological complete response (pCR or ypT0N0 stage), pathological downstaging (<ypT2N0 stage) and organ-confined disease (<ypT3N0 stage) at cystectomy.

Statistical methods

The diagram shown in Figure 1 issues the sample sizes for the analyses of the two endpoints discussed in the present study, i.e. renal impairment-free survival and pathological responses.

As regards descriptive results, qualitative data where reported as frequency and percentage, quantitative data as either means and standard deviation or as median and 95% range depending on the normal distribution assessment of the variable. Renal impairment-free survival was computed throughout chemotherapy with an event defined as a CrCl < 60 mL/min and a time of origin at D1 of the first cycle of chemotherapy. Patients with a clearance of creatinine < 60 mL/min at the baseline were excluded from this analysis. First, survival was computed by subgroups with Kaplan-Meier method. Curves are presented with 95% confidence interval adjusted with a Locally Weighted Regression and Smoothing Scatterplots (LOWESS) method. Then, covariates joint effects were analyzed in a Cox proportional hazard model with covariates backward selection on Akaike information criterion. Probabilities of complete response, pathological downstaging and organ-confined disease after NAC were analyzed on multivariate logistic models with covariates stepwise selection on Akaike information criterion. Considering the strong correlation between chemotherapy arm and the planned dose of cisplatin, an interaction term was introduced into these models, which made it possible to describe the effect of the dose of cisplatin in each arm. All statistical analysis where performed with R statistical software v3.6.2 (R Core Team (2020). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria).

Results

Patients' characteristics

Baseline characteristics of the 493 patients retained in the intent-to-treat analysis are reported in Table 1. Median age was 63 years (range, 33-80). According to body mass index (BMI), 40% and 19% of patients were classified as overweight or obese, respectively. Most patients were smokers or former smokers, 38% of patients had hypertension and 6.9% a history of coronary heart disease. Median CrCl was 87 mL/min (range, 51-152). Ten percent of patients had a calculated CrCl below 60 mL/min. Most patients received chemotherapy in the neoadjuvant setting (89% of cases). Characteristics were generally well balanced between the two arms, although the percentage of patients with diabetes was higher in the GC arm (p=0.026).

Cisplatin delivery

A total of 2,128 cycles of chemotherapy were delivered during the study, including 2,120 (99.6%) with cisplatin (Table 2). Thirty-one patients did not received cytotoxic doses according to their real body surface area (BSA) value since local investigators capped BSA at 2 m 2 in 25 patients, 2.1 m 2 in 1 patient and 2.2 m 2 in 5 patients. Full doses of cisplatin were given in 1866 (88%) cycles and 23 (4.7%) patients had to stop chemotherapy (12 GC and 11 dd-MVAC) because of renal failure. Five of them (22%) had a CrCl less than 60 mL/min at the onset of chemotherapy. In the GC arm, the median number of cycles was 4 regardless of the setting (neoadjuvant or adjuvant). However, the median total dose of cisplatin was significantly lower in patients who received adjuvant chemotherapy (p=0.002). In the dd-MVAC arm, the median numbers of cycles were 6 and 5 in the neoadjuvant and adjuvant setting, respectively, without any significant difference regarding the median total dose and dose intensity of cisplatin. CrCl between 60 and 90 retained significant prognostic value for the occurrence of renal impairment during chemotherapy (Table 3).

Changes in creatinine clearance during treatment

Impact of cisplatin delivery on pathological responses

Among 437 patients who received NAC, 397 of them (91%) underwent cystectomy.

Pathological complete response, pathological downstaging and organ-confined disease status were observed in 155 (39%), 224 (56%) and 278 (70%) patients, respectively. Baseline and treatment delivery characteristics were studied in multivariate logistic models to determine their potential role for each type of pathological responses (Table 4). Obese patients had a higher rate of complete pathological response. As previously reported, treatment arm had a significant, independent prognostic value with better efficacy of dd-MVAC regarding pathological downstaging and organ-confined disease status. No evidence for an additional effect of dose intensity on disease control was observed. In the GC arm, patients who received 4 cycles clearly achieved better outcomes as compared to patients who received 3 cycles or less for all types of pathological responses. In the dd-MVAC arm, increasing the number of cycles beyond four did not appear to provide statistically higher local control rates.

Discussion

Cisplatin has emerged as a major cytotoxic agent in urothelial tract tumors in the late 70s. [START_REF] Yagoda | Cisdichlorodiammineplatinum (II) in advanced bladder cancer[END_REF] As single agent, cisplatin was initially reported to produce response rates as high as 40%, but prospective randomized trials failed to confirm these results and complete responses were rarely observed. Although essentially palliative for patients with metastatic disease, the benefit of chemotherapy on response rates and overall survival was subsequently reported through the development of cisplatin-based combination regimens such as MVAC or CMV. [START_REF] Loehrer | A randomized comparison of cisplatin alone or in combination with methotrexate, vinblastine, and doxorubicin in patients with metastatic urothelial carcinoma: a Cooperative Group study[END_REF][START_REF] Mead | A randomized trial comparing methotrexate and vinblastine (MV) with cisplatin, methotrexate and vinblastine (CMV) in advanced transitional cell carcinoma: results and a report on prognostic factors in a Medical Research Council study[END_REF] Then their use in the perioperative setting led to establish NAC and radical cystectomy as current standard of care for MIBC. [START_REF] Grossman | Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer[END_REF][START_REF]Advanced Bladder Cancer Meta-analysis Collaboration. Neoadjuvant chemotherapy in invasive bladder cancer: update of a systematic review and meta-analysis of individual patient data[END_REF][START_REF]International collaboration of trialists. International phase III trial assessing neoadjuvant cisplatin, methotrexate, and vinblastine chemotherapy for muscleinvasive bladder cancer: long-term results of the BA06 30894 trial[END_REF] Given the potential for renal toxicity with cisplatin-based chemotherapy, adequate renal function is a prerequisite, with a common eligibility threshold of measured or calculated CrCl of ≥ 60 mL/min in clinical trials or medical oncologist community surveys. [START_REF] Galsky | A consensus definition of patients with metastatic urothelial carcinoma who are unfit for cisplatin-based chemotherapy[END_REF] However, no formal consensus has been established and robust data did not exist to support a specific renal function threshold and an optimal methodology to assess eligibility for as well as dose adaptations of cisplatin throughout therapy in MIBC. Regarding cisplatin eligibility, many contradictory results have been reported according to methods used for CrCl evaluation. In a retrospective study including 116 patients, the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation was more likely to deem a patient eligible for cisplatin compared with the CG formula, especially beyond 70 years of age. [START_REF] Tsao | Impact of the CDK-EPI equation for estimating renal function on eglibility for cisplatin-based chemotherapy in patients with urothelial cancer[END_REF] Another study found no difference between CDK-EPI equation, CG formula and the Modified Diet in Renal Disease (MDRD) equation, except for the subset of patients age > 80 in whom the CDK-EPI equation identified a much smaller proportion of cisplatin-eligible patients. [START_REF] Pal | CKD-EPI and Cockroft-Gault equations identify similar candidates for neoadjuvant chemotherapy in muscle-invasive bladder cancer[END_REF] Finally, in a third, retrospective study including 208 patients who received MVAC for advanced bladder cancer, concordance between calculated and measured CrCl was low, especially in those older than 65 years. The ability to complete at least 3 cycles of MVAC was related to a measured CrCl more than 60 mL/min, but not to a calculated CrCl more than 60 mL/min. [START_REF] Raj | Formulas calculating creatinine clairance are inadequate for determining eligibility for cisplatin-based chemotherapy in bladder cancer[END_REF] In the VESPER trial, the CG formula was used both for cisplatin eligibility (with 50 mL/min as threshold) and cisplatin dose adjustments throughout chemotherapy cycles. The present report confirms the relevance of this approach. Indeed, full doses of cisplatin were given in the majority of scheduled cycles (88%) and only 23 patients (4.7%) had to stop treatment because of renal failure. Moreover, 50 patients (10%) were included with a measured CrCl below 60 mL/min and only 10% of them had to stop chemotherapy because of renal failure. A trend towards poorer cisplatin delivery was observed in the adjuvant setting, with a lower median total dose of cisplatin in the GC arm and a reduced median number of cycles in the dd-MVAC arm. However, the number of patients who were treated in the adjuvant setting remains too small to draw firm conclusions. As the optimal number of cycles in the perioperative setting is still unknown, it is important to emphasize that increasing the number of cycles to six in the dd-MVAC arm did not lead to a clinically significant deterioration in renal function. Without major surprise, older age (≥ 70 years), low BMI (< 20 kg/m 2 ) and baseline CrCl < 90 mL/min were identified as independent prognostic variable for occurrence of renal impairment and therefore cisplatin dose modifications during chemotherapy.

In patients who are treated with NAC, achieving pCR in the bladder and lymph nodes at surgery has been shown to be a strong surrogate for survival outcomes. [START_REF] Grossman | Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer[END_REF][START_REF] Kassouf | P0 stage at radical cystectomy for bladder cancer is associated with improved outcome independent of traditional clinical risk factors[END_REF][START_REF] Sonpavde | Quality of pathologic response and surgery correlate with survival for patients with completely resected bladder cancer afer neoadjuvant chemotherapy[END_REF][START_REF] Petrelli | Correlation of pathologic complete response with survival after neoadjuvant chemotherapy in bladder cancer treated with cystectomy: A meta-analysis[END_REF] It has been also suggested that pathologic downstaging was associated with improved survival. [START_REF] Iyer | Neoadjuvant gemcitabine-cisplatin plus radical cystectomy-pelvic lymph node dissection for muscle-invasive bladder cancer: A 12year experience[END_REF][START_REF] Splinter | The prognostic value of the pathological response to combination chemotherapy before cystectomy in patients with invasive bladder cancer[END_REF] In the VESPER trial, we observed no significant different in the complete pathologic response rate between the two arms but higher pathologic downstaging and higher organ-confined disease rates in the dd-MVAC arm. [START_REF] Pfister | Randomized phase III of dose dense methotrexate, vinblastine, doxorubicin and cisplatin (dd-MVAC) or gemcitabine and cisplatin (GC) as peri-operative chemotherapy for patients with muscle-invasive bladder cancer (MIBC). Analysis of the GETUG/AFU V05 VESPER trial secondary end-points: chemotherapy toxicity and pathological responses[END_REF] Our prospective randomized data offered the possibility to refine the potential relationship between patients' baseline characteristics, cisplatin delivery and pathologic responses. BMI ≥ 25 kg/m 2 and a minimum number of cycles of 4 cycles of chemotherapy were identified as independent prognostic variables regarding pCR at surgery. The influence of BMI for patients treated with NAC for MIBC was recently studied in a retrospective, single institution experience. [START_REF] Belaube | Neoadjuvant chemotherapy for urothelial muscle-invasive bladder cancer: influence of body mass index on efficacy and toxicity[END_REF] Toxicity and efficacy of NAC were compared between obese patients receiving chemotherapy based on capping at a body-surface area of 2.2 m 2 and healthy weight patients. Obese patients were individually paired with normal BMI patients sharing similar clinical and biological characteristics. The pCR rate was similar in both groups. In the VESPER trial, 31 (6.3%) patients did not receive optimal cytotoxic doses since BSA was capped by local investigators. Therefore, a lower pCR rate related to a potential drug underdosing could have been expected in obese patients. The opposite observation might be related to sarcopenic obesity. However, sarcopenia has previously been linked to renal impairment and prognosis after NAC, [START_REF] Lyon | Sarcopenia and response to neoadjuvant chemotherapy for muscle-invasive bladder cancer[END_REF][START_REF] Régnier | Impact of sarcopenia status of muscle-invasive bladder cancer patients on kidney function after neoadjuvant chemotherapy and post cystectomy complications[END_REF] but not to pathological response. [START_REF] Lyon | Sarcopenia and response to neoadjuvant chemotherapy for muscle-invasive bladder cancer[END_REF][START_REF] Stangl-Kremser | Sarcopenia as a predictive factor for response to upfront cisplatin-based chemotherapy in patients with muscle-invasive urothelial bladder cancer[END_REF] Moreover, we did not observe a higher myelosuppression suggesting greater exposure due to weight-based dose calculation in obese patients.

The optimal number of NAC cycles remains undefined. A survival benefit for 3 cycles of MVAC or CMV is supported by level 1 evidence. [START_REF] Grossman | Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer[END_REF][START_REF]Advanced Bladder Cancer Meta-analysis Collaboration. Neoadjuvant chemotherapy in invasive bladder cancer: update of a systematic review and meta-analysis of individual patient data[END_REF][START_REF]International collaboration of trialists. International phase III trial assessing neoadjuvant cisplatin, methotrexate, and vinblastine chemotherapy for muscleinvasive bladder cancer: long-term results of the BA06 30894 trial[END_REF] Alternative combinations with 3 to 4 cycles of dd-MVAC were reported in 2 phase II trials. [START_REF] Choueiri | Neoadjuvant dose-dense methotrexate, vinblastine, doxorubicin and cisplatin with pegfilgrastim support in muscle invasive urothelial cancer: Pathologic, radiologic and biomarker correlates[END_REF][START_REF] Plimack | Accelerated methotrexate, vinblastine, doxorubicin and cisplatin is safe, effective and efficient neoadjuvant treatment for muscle invasive bladder cancer. Results of a multicenter phase II study with molecular correlates of response and toxicity[END_REF] The pCR rates (26% and 38% after 4 and 3 cycles, respectively) were roughly similar to that observed in the VESPER trial (42%). Our data strongly argue for a lesser chance of obtaining a pCR when less than 4 cycles of GC or dd-MVAC are delivered. However, increasing the number of cycles beyond four did not appear to provide statistically higher local control rates.

In conclusion, prospective data from the VESPER trial provide answers as to the optimal number of cisplatin-based chemotherapy cycles in the neoadjuvant setting for patients with MIBC. A minimum number of 4 cycles is required to optimize the chances of getting a pCR at cystectomy after NAC. Increasing the number to 6 cycles does not lead to a clinically significant deterioration in renal function but the benefit on patients' survival remains to be proven. Covariates age, sex, OMS status, cT tumor stage, tobacco, diabetes were excluded from each final model with the stepwise selection. Chemotherapy arm and its interaction with total dose of cisplatin were forced in the selections.

Figure 2 -Evolution of median creatinine clearance from baseline to the end of chemotherapy

Whole patient sample (A), According to treatment arms (B) According to baseline creatinine clearance (C). For A and B, the q1-q3 interval is presented with a colored area. For each curve, the number of patients assessed at the baseline is indicated, then for each cycle the percentage of these patients whose creatinine clearance was evaluated.

Figure 1

 1 Figure 1 underlines serial changes in CrCl during chemotherapy in the whole population and

Figure 3 -

 3 Figure 3 -Renal impairment-free survival curves Kaplan-Meier curves with 95% confidence area according to treatment arms (A, N=493) and according to baseline creatinine clearance (B, N=443, patients with creatinine clearance < 60 mL/min at baseline are not presented).

Table 1 -Baseline patients' characteristics

 1

	Variable	GC	dd-MVAC	Total
		(N=245)	(N=248)	(N=493)
	Age, years, No (%)
	<60	75 (31)	82 (33)	157 (32)
	60-69	119 (49)	124 (50)	243 (49)
	≥70	51 (21)	42 (17)	93 (19)
	Mean (standard deviation)	63 (7.6)	62.6 (7.9)	62.8 (7.7)
	Sex, No (%)
	Male	206 (84)	202 (81)	408 (83)
	Female	39 (16)	46 (19)	85 (17)
	Body mass index, kg/m 2 , No (%)
	<18.5	3 (1.2)	9 (3.6)	12 (2.4)
	18.5-<25	95 (39)	95 (38)	190 (39)
	25-<30	100 (41)	99 (40)	199 (40)
	≥30	47 (19)	45 (18)	92 (19)
	Mean (standard deviation)	26.6 (4.7)	26.0 (4.4)	26.3 (4.5)
	Body surface area (m 2 )
	Mean (standard deviation)	1.9 (0.2)	1.9 (0.2)	1.9 (0.2)
	Medical history, No (%)
	Tobacco	198 (84)	197 (81)	395 (80)
	Hypertension	100 (41)	89 (36)	189 (38)
	Coronary artery disease	13 (5.3)	19 (7.7)	32 (6.5)
	Diabetes	14 (5.7)	4 (2)	18 (4)
	Performance status, No (%)
	0	171 (70)	165 (67)	336 (68)
	1	72 (29)	82 (33)	154 (31)
	Unknown	2 (0.8)	1 (0.4)	3 (0.6)
	Hemoglobin (g/dL)
	Median (95% range)	14.1 (10.7-16.5) 14.0 (11.0-16.5) 14.0 (10.8-16.5)
	Alkaline phosphatases (UI/L)
	Median (95% range)	73 (46-138)	73 (42-148)	73 (45-146)
	Total bilirubin (mg/L)
	Median (95% range)	4.4 (1.8-11.0)	4.7 (2.0-11.0)	4.7 (1.8-11)
	Serum creatinine (mg/L)
	Median (95% range)	9 (6-14)	9 (5-14)	9 (5-14)
	Creatinine clearance, mL/min, No (%)
	50-59	27 (11)	23 (9.3)	50 (10)
	60-89	113 (46)	107 (43)	220 (45)
	≥90	105 (43)	118 (48)	223 (45)
	Median (95% range)	86 (52-157)	88.5 (51-147)	87 (51-152)

Table 2 -Cisplatin delivery

 2

		Neoadjuvant chemotherapy	Adjuvant chemotherapy
		GC	dd-MVAC	GC	dd-MVAC
		(N=219)	(N=218)	(N=26)	N=(30)
	Number of cycles
	Median (range)	4 (1-4)	6 (0-6)	4 (1-4)	5 (1-6)
	Total	803	1093	92	140
	Total with cisplatin	803	1087	91	139
	Total with full doses of cisplatin (%)	745 (93)	926 (85)	73 (80)	122 (88)
	Total dose (mg)	522 (134-608)	700 (132-895)	454 (125-579)	675 (131-843)
	Median (95% range)
	Dose intensity (mg/m 2 /week)	22.9 (16.9-24.1) 31.8 (20.5-35.7) 21.9 (14.6-23.8) 33.8 (21.5-35.2)
	Median (95% range)
	Relative dose intensity	98%	91%	94%	97%

Table 3 -Cox proportional hazard model for renal impairment during chemotherapy

 3

	Baseline characteristics	Hazard ratio (95% CI)	p value
	Age (years)
	< 60	-
	60-69	2.37 (1.21-4.64)	0.012
	≥ 70	4.85 (2.27-10.4)	< 0.001
	Body mass index (kg/m 2 )
	< 20	3.20 (1.43-7.15)	0.005
	20-<25	-
	25-<30	0.82 (0.48-1.41)	0.5
	≥ 30	0.70 (0.35-1.42)	0.3
	Coronary artery disease
	No	-
	Yes	1.82 (0.86-3.86)	0.12
	Creatinine clearance (mL/min)
	≥ 90	-
	60-89	2.93 (1.69-5.10)	< 0.001
	Chemotherapy arm
	GC	-
	dd-MVAC	1.20 (0.75-1.93)	0.5

Covariates tobacco, timing of chemotherapy (adjuvant or neoadjuvant), sex, lymph nodes involvement, diabetes, high blood pressure and OMS status were excluded in this order from the final model with the backward selection. Chemotherapy arm was forced in the selection.

Table 4 -Logistic model for pathological responses at cystectomy

 4

		N	Complete pathological	Pathological downstaging	Organ-confined disease
			response
			Odd ratio (95% CI) p value	Odd ratio (95% CI) p value	Odd ratio (95% CI) p value
	Body mass index
	(kg/m 2 )
	< 20	23	0.83 (0.30-2.29)	0.7	0.41 (0.16-1.10)	0.078
	20-<25	126	-		-
	25-<30	164	1.90 (1.16-3.12)	0.011	1.67 (1.03-2.73)	0.038
	≥ 30	84	1.83 (1.02-3.29)	0.043	1.29 (0.72-2.30)	0.4
	Creatinine clearance
	at baseline (mL/min)
	≥90	187					-
	60-89	174					0.64 (0.39-1.04)	0.069
	50-59	36					0.28 (0.13-0.60)	0.001
	High blood pressure
	No	237					-
	Yes	160					0.65 (0.40-1.03)	0.065
	Coronary artery disease
	No	369			-
	Yes	28			2.05 (0.83-5.02)	0.12
	Chemotherapy arm
	GC	198	-		-		-
	dd-MVAC	199	1.40 (0.77-2.56)	0.3	2.06 (1.08-3.93)	0.029	2.46 (1.10-5.52)	0.028
	Cisplatin dose-intensity
	(mg/m 2 /week)
	≥ 30	218	-		-		-
	25-30	46	1.04 (0.50-2.18)	0.9	0.71 (0.33-1.51)	0.4	0.75 (0.31-1.79)	0.5
	< 25	132	1.06 (0.37-3.02)	0.9	0.84 (0.30-2.39)	0.7	0.75 (0.22-2.60)	0.7
	Total dose of cisplatin
	(mg/m 2 )
	GC arm
	≥ 270 (4 cycles)	134	-		-		-
	< 270	64	0.46 (0.23-0.89)	0.022	0.49 (0.26-0.91)	0.024	0.47 (0.25-0.88)	0.019
	dd-MVAC arm
	≥ 410 (6 cycles)	65	-		-		-
	340 to 410 (5 cycles)	62	0.56 (0.27-1.15)	0.12	0.61 (0.28-1.29)	0.19	0.89 (0.33-2.37)	0.8
	270-340 (4 cycles)	38	0.85 (0.38-1.93)	0.7	0.67 (0.28-1.59)	0.4	0.44 (0.16-1.20)	0.11
	< 270 (< 4 cycles)	34	0.67 (0.28-1.58)	0.4	0.55 (0.23-1.32)	0.18	0.39 (0.14-1.10)	0.074

Acknowledgements

The authors strongly thank Cécile Pourcher, Frédérique Caillot and Assia Ferrane, Rouen University Hospital Clinical Research Assistants, for their active participation in the multicenter monitoring of this phase III trial. The Northwest Data Center (CTD-CNO) is acknowledged for managing the data. It is supported by grants from the French National League against Cancer (LNC) and the French National Cancer Institute (INCa). We also thank all urologists who were involved in the trial: G. Pignot (Marseille), J.P. Fendler (Lyon), M. Soulié (Toulouse), L. Guy (Clermont-Ferrand), G. Verhoest (Rennes), N. Mottet (Saint-Etienne), A. Doerfler (Caen), A.R. Azzouzi (Angers), P. Mongiat-Artus (Paris, Saint-Louis), P. Eschwege (Nancy), J.L. Hoepffner (Bordeaux), H. Lang (Strasbourg), E. Mandron (Le Mans), A. Colau (Paris, Diaconesses), A de La Taille (Créteil), F. Kleinclauss (Besançon), J. Irani (Villejuif), S. Larre (Reims), M. Zerbib (Cochin), J. Rigaud (Nantes), S. Droupy (Nimes), M. Roupret (Paris), C. Legon (La Seyne sur Mer), N. Letang (Perpignan), N. Gaschignard (La Roche sur Yon), J.L. Davin (Avignon) .

Funding

The GETUG/AFU V05 VESPER randomized phase III study was supported by a grant from the French Ministry of Health (PHRC 2011-037).

Sponsor

The sponsor of the study was the University Hospital in Rouen, France.

Disclosure

Authors have declared no conflicts of interest.


J Gautron
email: joel.gautron@inrae.fr

S Réhault-Godbert

T G H Van De Braak

I C Dunn

Review: What are the challenges facing the table egg industry in the next decades and what can be done to address them?

Keywords: Egg production, Hen housing, sustainable farming, dual purpose, in-ovo sexing Implications

There has been a strong consumer demand to take welfare into account in animal production, including table eggs. This is particularly true in Europe and North America but increasingly around the world. We review the main demands that are facing the egg industry driven by economic, societal and sustainability goals. We describe solutions already delivered by research and those that will be needed for the future.

Already table egg consumption patterns have seen a major shift from cage to noncage production systems because of societal pressures. These often feature free range and organic production. These changes likely signal the future direction for the layer sector with the acceleration of the conversion of cage to barn and aviary systems with outdoor access. This can come with unintended consequences from bone fracture to increased disease exposure, all requiring solutions. In the near future, the laying period of hens will be routinely extended to improve the economics and environmental footprint of production. Many flocks already produce close to 500

Introduction

Egg production is a good example of the major changes that have occurred in the agricultural sector in response to changing social demands. There is currently strong consumer pressure for the consumption of healthy, high-quality animal products that take into account animal welfare and sustainability. The consideration of the ethical dimension in this sector has resulted in many examples of major changes to the way that eggs are produced, in order to respond to societal demands. The main changes to the production system concern the gradual abandonment of cage-housing systems, the demand to not kill male chicks and the lengthening of the production period. The latter will result in fewer birds being slaughtered each year and fewer hens being required throughout the whole production system, both in breeding and in production, to produce the same quantity of eggs. As a result, this method of production should have less negative impact in terms of the environment and use of resources. This may balance some of the negative impacts of reduced efficiency resulting from other adaptations discussed. All these developments and trends will have a major impact on the poultry sector, and will shape table and fertilized egg production in the coming years. The objective of this review was therefore, 1) to describe the issues facing the egg production chain and how the industry may evolve and 2) to examine existing scientific research to address these issues, indicating the current state of the art in the production of eggs for human consumption and future innovations in the industry. It will finally give examples of initiatives towards more ethical animal husbandry.

Evolution of the system of egg production from cage to non-cage systems

Current production methods in Europe are in line with the five freedoms of animal welfare: freedom from hunger and thirst, from discomfort, from pain injury or disease, to express normal behaviour and freedom from fear and distress [START_REF] Efsa | Welfare aspects of various systems for keeping laying hens[END_REF].

However, among the four authorised modes of production (organic, free range, barn and enriched cages), there is a growing consumer mistrust of eggs produced by enriched-caged hens even though the traditional barren cage was banned since 01/01/2012 (Council Directive 1999/74/EC). The enriched or furnished cages that replaced these classical cages, favor the expression of more natural behaviours by the hens but this positive change has not been readily understood by consumers.

Furthermore, these changes have not solved all behavioural issues of cages as highlighted by the EFSA (2005) report on laying hens' welfare. Indeed, litter supply in furnished cages is still a major issue, making the hens unable to show normal foraging and dustbathing behaviour [START_REF] Efsa | Welfare aspects of various systems for keeping laying hens[END_REF]. This directive has resulted in a strong segmentation of the markets. In 1996, non-cage systems accounted for 8% of the EU laying hen population, 30% in 2009, 46% in 2017 and 51% in 2019 (80% of laying hens were in cages in 2003, 49% in 2019) (ITAVI 2019) (figure 1). The proportion of hens reared in non-cage systems is currently increasing sharply, although it remains very heterogeneous in Europe (from less than 10% in Spain and Poland to more than 85% in the Netherlands, Germany and Austria) (Figure 1). Like many of Europe's member states, French production has become increasingly diversified since the late 1990s. The number of laying hens in non-cage systems was 19% in 2008 and has reached 46% in 2019 (Figure 1) [START_REF] Itavi | Situation du marché des oeufs et des ovoproduits : Edition mars[END_REF]. In contrast, the egg consumption by distribution channels did not change during the last decade (Figure 2), with about 60% of eggs consumed as shelled eggs and about 40 % as egg products in France [START_REF] Itavi | Situation du marché des oeufs et des ovoproduits : Edition mars[END_REF]. The supermarkets and hypermarkets in France, attentive to campaigns from non-governmental organizations on the welfare of laying hens, have announced an end to the marketing of eggs from cage systems within the next 2-5 years (Table 1). On 18th February 2018, the French Agriculture Minister Stéphane Travert confirmed that the government would work on a ban of table eggs from cage systems by 2022 for whole egg sales [START_REF] Tassard | Stéphane Travert confirme la fin de l'élevage en batterie pour la production d'oeufs-coquilles[END_REF]. The French mediator for agricultural trade relations, in his 2017 report, points out that "the refusal to market eggs produced by caged hens ... is on the way to become the norm regardless of any regulatory developments".

However, the objective of converting the existing furnished cage systems to non-cage systems is not economically or materially feasible by the end of 2020 and even 2025 according to the mediator of the French republic and ITAVI economic studies. As a result, there will be disruptions in supply and a significant increase in intra-European imports. For example, Germany has eliminated cages in favour of floor-based production, either in indoor or free-range systems, and is in a position to export.

Despite the fact that Great Britain has left the European Community, a similar trend can be observed. There are four recognised forms of laying hen production following EU definitions, three non-cage systems and the furnished cage. The three non-cage production systems are Free-Range, organic and Barn egg production. Organic egg production must also be free range, but follows different standards notably in terms of stocking density, the origin of the animal feed, medication and beak trimming; Barn egg production (code 2), which is similar to free range in terms of housing, but with the notable exception that the hens do not have access to the outdoors. In the UK in 2019, there was around 2%, 3%, 53% and 42% of eggs packed from barn, organic, free range and enriched cages systems, respectively (DEFRA, 2020). Both organic and barn systems are increasing, but from a low starting point. It is believed that, as retailers commit to going cage free, barn production will be increased to give a lowprice alternative to free range eggs, although with the lack of consumer understanding of barn production that may be a challenge [START_REF] Porter | The new standard has been agreed upon, but there's a hefty price tag attached for producers[END_REF]. Indeed, as in France, some retailers have already said that they will not sell barn eggs [START_REF] White | Sainsbury's set to stop selling barn eggs in[END_REF]. Much of the egg production in the UK (~90%) is part of the Lion Quality Code of Practice that has enhanced requirements for welfare in terms of stocking densities for free range hens, nest box space, lighting and the handling of end-of-lay hens. It also focuses heavily on egg hygiene and microbiological quality, which was the initial impetus for its creation. This Code of Practice can be a major driver for change if its promoters choose to adopt new standards in terms of welfare. For the free-range sector, research will continue on how to design the indoor environment to better allow natural behaviour but reduce damage from collisions [START_REF] Stratmann | Modification of aviary design reduces incidence of falls, collisions and keel bone damage in laying hens[END_REF].

Initiatives to tackle the problems of injurious pecking are also underway [START_REF] Nicol | The prevention and control of feather pecking: application to commercial systems[END_REF]. Another important issue is the way to reduce poultry pandemics and others diseases that can be transmitted to humans. Breeding systems with outdoor access increase the risk of exposure to wild animals including other birds. The recent COVID-19 pandemic, where wild animals are suspected to be the reservoir of virus, has reinforced this concern, although there is no evidence of birds being involved in the transmission in this case. Poultry pandemics (like Avian Influenza), raise the question of the relocation of bird farms producing eggs from high-risk areas to areas that are less prone for migrating birds carrying Avian Influenza (ANSES 2021).

Besides the improvement of animal welfare, the sustainability aspects of the egg industry should be further investigated to improve housing systems with respect to the needs of the laying hens, but also to lower the ecological footprint of egg production. They should permit the use of more by-products, residual waste, to improve circular egg farming [START_REF] De Olde | Sustainability challenges and innovations in the Dutch egg sector[END_REF]. Overall can new systems be developed that have benefits for the hen that do not have negative welfare consequences and are more sustainable? Perhaps the greatest challenge will be: can the consumer be educated to accept different systems which are more sustainable?

Extending the production period

Currently, laying hens start laying at around 18 weeks of age and peak laying is around 25 weeks of age. Eggs are well valued from the moment that the weight of the egg has reached a threshold value (e.g. 53 g in EU and 49.3 g in USA). The majority of the flocks are depopulated when the laying rate is reducing and, most importantly the incidence of downgraded eggs increases to unprofitable levels. At this point there are still many saleable eggs but the high variability in egg quality means a threshold is reached where less than about 75% of the eggs are marketable, although the threshold can vary depending on prevailing economics. In 2016, this often occurred at around 72 weeks in free-range flocks in the UK, i.e. after one year of production [START_REF] Bain | Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?[END_REF], but already a Belgian study indicated that the end of production could vary from 74 to 92 weeks, with an egg laying rate of 79% [START_REF] Molnar | Changes in egg quality traits during the last phase of production: is there potential for an extended laying cycle?[END_REF]. Reproduction in birds is controlled by the hypothalamus according to different environmental and endocrine stimuli. As the hen ages, environmental stimuli that were previously stimulatory no longer have that effect and potential inhibitory factors may increase, which results in reduced hypothalamic activity of the cells driving reproduction [START_REF] Dunn | Endocrinology and Genetics of the Hypothalamic-Pituitary-Gonadal Axis[END_REF]). The consequence is a loss of weight and functionality of the oviduct, which leads to an increase in the number of days of rest (without laying) and defective eggs [START_REF] Solomon | The oviduct in chaos[END_REF]. However, selection for egg production has successfully reduced the effects of age on the reproductive system, with the result of reducing the amount of variation in egg production rate towards the end of the traditional laying period. Essentially the majority of hens were maintaining production of one egg a day. To perform selection and improve the sustainability of the industry, poultry breeders have extended the laying periods of their pure line birds to make variation visible (pers. comm. Teun van de Braak). It is now possible to select those individuals that are observed in older flocks that can maintain high oviposition rates with good shell quality [START_REF] Molnar | Changes in egg quality traits during the last phase of production: is there potential for an extended laying cycle?[END_REF]. Accessing this variation has allowed an increase in the persistence of egg production and maintenance of egg quality at an advanced age [START_REF] Bain | Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?[END_REF].

Breeders indicated 10 years ago, that they wanted to select strains of laying hens capable of laying sustainability up to 100 weeks of age, for a total production of almost 500 eggs per birds by 2020 [START_REF] Bain | Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?[END_REF], which has been achieved. [START_REF] Bain | Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?[END_REF] indicate that an additional production of 25 eggs per hen could potentially reduce the laying hen flock in Great Britain by 2.5 million hens. This cumulative effect is obtained as shown in figure 3, due to the pyramidal structure of production in the sector. An increase of 10 weeks of production would preserve 1 g of potentially polluting nitrogen per dozen eggs produced [START_REF] Molnar | Changes in egg quality traits during the last phase of production: is there potential for an extended laying cycle?[END_REF].

However, any improvement in egg-laying persistence must be achieved with consistent egg quality. Egg weight increases by 70 mg per week between 60 and 80 weeks of age [START_REF] Molnar | Changes in egg quality traits during the last phase of production: is there potential for an extended laying cycle?[END_REF]. While the other egg constituents remain constant, there is a decrease in shell thickness (-0.23 µm per week) and a decrease in breaking strength with increasing age of the hen. Haugh units are an indicator of egg white quality. They measure the height of the egg white after breaking the egg, which is related to the viscosity of the colloidal gel of the egg white. This gel-like structure limits bacterial proliferation and migration towards the yolk and is essential for maintaining the hygienic quality of the egg. However, Haugh units reduce over the course of the production period [START_REF] Whitehead | Overview of bone biology in the egg-laying hen[END_REF][START_REF] Bain | Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?[END_REF][START_REF] Molnar | Changes in egg quality traits during the last phase of production: is there potential for an extended laying cycle?[END_REF]). There may be other consequences of keeping hens for longer, like moulting that should result in an absence of eggs during several weeks and an economic loss because hens are still fed while they do not produce eggs. It is essential to maintain good bone quality in the laying hen, especially when the hen is getting older. A laying hen requires between 2 and 2.5 g of calcium daily for the production of an eggshell. About 2/3 of this calcium is provided directly by the feed, the remaining 1/3 comes from storage by demineralisation of the medullary bone. The calcium from the medullary bone is necessary in the second part of the shell mineralisation process. This occurs at night, when the hen has no access to feed although birds do store food in the crop for several hours. Medullary bone is capable of rapid absorption and renewal [START_REF] Whitehead | Overview of bone biology in the egg-laying hen[END_REF]), which can be optimised by dietary calcium sources (content, quality and particle size). Even with a perfectly controlled diet, bone demineralization is a natural phenomenon that can also affect the structural bone, ultimately leading to osteoporosis. This pathology, which can be prevalent in old hens, leads to bone fragility and keel bone fractures that severely impact the welfare of laying hens [START_REF] Armstrong | Keel bone fractures induce a depressive-like state in laying hens[END_REF]. Bone quality and fractures in these hens are currently a major issue in the table egg sector [START_REF] Sandilands | The laying hen and bone fractures[END_REF]. It is easy to observe hens in a flock with very fragile or very strong eggs and the same applies to bones. However, there is relatively little knowledge about whether hens with bone defects are those that lay eggs with fragile or strong shells.

In studies where genetic correlations between egg quality and bone quality have been examined, there is little evidence that the two traits are linked, with only the keel bone density in one line being significantly correlated with egg breaking strength [START_REF] Dunn | No evidence that selection for egg production persistency causes loss of bone quality in laying hens[END_REF]. Whilst there were no significant associations for the tibia or humerus strength or density with egg quality, there was a genetic correlation with onset of lay and early egg number, but only in one line. Although not significant, there was a suggestion that genetic loci explaining variation in egg quality might be present at the same loci as one for bone quality [START_REF] Dunn | Lack of genetic correlation between bone quality and egg number in the laying hen suggests increased egg production may not be responsible for reduction in bone quality[END_REF] but in a related study, lines of hen successfully selected to have differences in bone strength did not have differences in egg quality [START_REF] Fleming | Relationships between genetic, environmental and nutritional factors influencing osteoporosis in laying hens[END_REF]. This is not to say that egg laying is not related to issues of bone quality in laying hens, if hens do not lay eggs at all they have better bone quality [START_REF] Eusemann | The Role of Egg Production in the Etiology of Keel Bone Damage in Laying Hens[END_REF]). However, the quality of the egg or the persistency of lay do not seem to be critical components in determining bone quality of a hen. Rather, it is possible that the onset of lay, which is intrinsically related to the body weight of the hen and the genetic factors that directly affect bone quality, appear to be most important [START_REF] Dunn | Lack of genetic correlation between bone quality and egg number in the laying hen suggests increased egg production may not be responsible for reduction in bone quality[END_REF]. Research is ongoing to resolve some of the questions on the relationship between persistent egg production and bone quality and other welfare issues, and how the physiology, nutrition and welfare of the older hen will be affected. Programmes conducted in partnership with researchers in the sector are underway to ensure best practice which will help support the longer laying period [START_REF] Toscano | Explanations for keel bone fractures in laying hens: are there explanations in addition to elevated egg production?[END_REF].

Prolonging the laying cycle can only be achieved when the health of the birds remains in good condition. For the average flock, weekly mortality figures tend to increase when the birds are getting older. Continuous genetic selection for improved livability and improved knowledge on nutrition and flock management have resulted in lower weekly mortality numbers. There is some optimism that, in a similar way that selection for saleable egg production reduces the incidence of egg defects [START_REF] Wolc | Genetic parameters of egg defects and egg quality in layer chickens[END_REF], health issues will be reduced by selection for saleable eggs as negative health traits often result in reduced egg production. As the genetics for bone quality suggests, more emphasis should be placed on the rearing period. The mindset is changing from seeing it as a period of costs to a period of investment in the bird's productive achievements later on in life. For decades, birds have been selected to come into lay at an earlier age. From the overview of 37 tests of a North Carolina random sample and subsequent layer performance and management tests [START_REF] Anderson | Changes in commercial laying stock performance, 1958-2011: thirty-seven flocks of the North Carolina random sample and subsequent layer performance and management tests[END_REF], it can be observed that the age at sexual maturity has been reduced by more than a month. When we look at the last, 10 tests and talking to breeding companies (communication with Hendrix Genetics), it can be observed that selection is not continuing for birds to come into lay even earlier. The long-life layer needs sufficient time to grow and develop. Bodyweights at week 6 and 17 of the rearing period are associated with productivity later in in the bird's life, i.e. higher bodyweights at the crucial development stages are positively associated with higher peaks of production, higher egg weights and improved persistency in egg production and, as discussed previously in connection with bone quality (communication with Hendrix Genetics) (table 2).

The accelerated change in housing systems and the prohibitions on management practices such as beak trimming in several EU countries (Austria, Germany, Netherlands, etc.) is the most recent manifestation of a trend that has resulted in changes in selection criteria for laying hens. The traditional approach that was merely focused on the economic aspects of egg production has shifted. Today, breeding programs and selection indexes include more poultry health and welfare traits than ever before. Next to relatively well investigated traits such as livability, bone strength, disease resistance and feather cover, new traits related to behavior, such as negative social interactions between birds, and behavior in cage free housing systems have been adopted by the breeding companies [START_REF] Brinker | The genetic architecture of socially-affected traits: a GWAS for direct and indirect genetic effects on survival time in laying hens showing cannibalism[END_REF]).

Not only have health and welfare received increased attention, breeding companies are showing their commitment and making their contribution to set the standard for sustainable egg production. The environmental impact per kilogram of eggs produced has significantly decreased during the past decades. The review by [START_REF] Pelletier | Comparison of the environmental footprint of the egg industry in the United States in 1960 and 2010[END_REF] showed a comparison of the environmental footprint of the egg industry in the United States in 1960 and 2010. They showed an enormous reduction in the environmental footprint per kilogram of eggs produced: the environmental footprint for 2010 was 65% lower in acidifying emissions, 71% lower in eutrophying emissions, 71% lower in greenhouse gas emissions, and 31% lower in cumulative energy demand compared to 1960. Despite the 30% higher table egg production in 2010, the total environmental footprint was significantly lower compared to 1960. Pelletier at al (2014) estimated that 28 to 43% of this improvement in lower environmental footprint could be attributed to the improvements in the performance of the birds. Abín et al (2018) used a Spanish case study for an environmental assessment of intensive egg production. Their study showed that after the production of the hen feed, the purchase of new laying hens to replace the old flock contributed most to the harmful environmental impact of intensive egg production. By extending the productive lifetime of the laying hens in the breeding pyramid and not just at the production level, the environmental impact could be further reduced, as fewer replacements flocks are needed.

Alternatives to the culling of one-day-old male chicks

A major issue that has raised ethical concerns within the poultry sector is the fate of male chicks from laying strains. Although chickens can be used for both meat production and egg production, there is a trade off between the two traits (Wolc et al. 2012, Giersberg and[START_REF] Giersberg | Rearing Male Layer Chickens: A German Perspective[END_REF]. As a result, there has been specialised breeding of chickens for either egg production or for meat production [START_REF] Leenstra | Layer breeding programmes in changing production environments: a historic perspective[END_REF][START_REF] Sakomura | Modeling egg production as a means of optimizing dietary nutrient contents for laying hens[END_REF]). These strains of hens are very efficient at either only egg production or only meat production, but do not compete economically in each other's market. Hens selected to produce eggs for human consumption have a low body weight of around 1.7kg that is reached at around 20 weeks of age (sexual maturity), where they start to convert their feed into egg nutrients very efficiently [START_REF] Ahammed | Comparison of Aviary, Barn and Conventional Cage Raising of Chickens on Laying Performance and Egg Quality[END_REF][START_REF] Bain | Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?[END_REF]). Hens at the end of lay are used for human consumption, but the males have limited added-value as they grow slowly and the live weight and meat yield on the carcase do not meet the meat quality/yield criteria to be marketed [START_REF] Giersberg | Rearing Male Layer Chickens: A German Perspective[END_REF]. As only the females lay eggs, half of the hatched chicks are therefore non-marketable males. Day-old chicks are sexed at hatch using cloacal or vent sexing, or via sex linked feather features (feather color or covert length). Only a limited number of males are bred to allow the reproduction of future offspring. As a result, billions of male chicks have no commercial value [START_REF] Weissmann | Sexing domestic chicken before hatch: a new method for in ovo gender identification[END_REF][START_REF] Galli | In Ovo Sexing of Domestic Chicken Eggs by Raman Spectroscopy[END_REF][START_REF] Giersberg | Rearing Male Layer Chickens: A German Perspective[END_REF], and are culled rapidly after hatch by asphyxiation or maceration. These authorized practices elicit legitimate questions in terms of animal welfare and the ethics of hatching eggs without an agricultural output. The joint announcement by the French and German ministers to ban the culling of one-day old male chicks by the end of 2021 has given some urgency to finding solutions and several methods are close to market while some of them are being used by hatcheries. The three main alternative approaches to the practice of killing male chicks are; 1) to identify mechanisms that can, ideally completely, imbalance the sex ratio in favor of females, 2) to develop tools that would allow the determination of the sex of the embryo in ovo prior to hatch, 3) to develop dual-purpose strains where female chicks would be reared as future egg-laying hens and male chicks for meat. Ultimately the systems developed will also need to be acceptable to consumers.

Skewing primary sex-ratio or hatching sex-ratio

It is well-known that the primary avian sex-ratio can be affected by environmental factors such as diet, physiology, hormonal status and conditions as well as the genetic background. However, the effects are relatively modest and can change over the lifetime of a hen [START_REF] Klein | Primary sex ratio in fertilized chicken eggs (Gallus gallus domesticus) depends on reproductive age and selection[END_REF]. Thus, it is quite difficult to know how sex ratios could be reliably altered by poultry breeders using current knowledge without gene editing or transgenesis. Research into the basic mechanisms of sex determination in birds certainly continues to be warranted. Another level of complexity is that the primary sex-ratio of fertilized eggs does not necessarily reflect the sex ratio at hatch, which suggests that a sex-dependant selection during incubation depending on the genetic background and age of laying hens may occur [START_REF] Klein | Primary sex ratio in fertilized chicken eggs (Gallus gallus domesticus) depends on reproductive age and selection[END_REF]. The Israeli company SOOS [START_REF] Soos | Egg sex determination[END_REF]) used this concept to develop an incubation system combining different incubation parameters that seem to reverse males into female chicks. According to their claims, resulting animals can efficiently lay eggs at sexual maturity. However, to date, scientific information about the underlying mechanisms and short/long term impacts on feminized chickens are missing and need to be addressed before this approach can be validated by the scientific community and, ultimately, authorities and society. In the light of this, it seems necessary to develop other strategies until realistic methods to skew the primary sex ratio are available.

Sexing eggs

The second strategy is to detect and discard male eggs before hatching instead of killing one-day-old male chicks [START_REF] Krautwald-Junghanns | Current approaches to avoid the culling of day-old male chicks in the layer industry, with special reference to spectroscopic methods[END_REF]). This approach relies on the development of on-ovo or in-ovo sexing methods that are based on the detection of sexual dimorphic traits or molecules. Several approaches have been tried during the last decade to obtain a method that can be used in practice in hatcheries. There are many prerequisites to develop an operational sexing method than can be effective at an industrial level. The analysis must be rapid, inexpensive, highly accurate and have no impact on chick hatching rate, health and performance [START_REF] Kaleta | Approaches to determine the sex prior to and after incubation of chicken eggs and of day-old chicks[END_REF]. There is also one other constraint. Ovosexing methods as an alternative to the culling of one day-old male chicks will need to meet the required social/consumer acceptability [START_REF] Gremmen | A Public Survey on Handling Male Chicks in the Dutch Egg Sector[END_REF]. Consumers may not differentiate the killing of an embryo from the killing of a day-old chick. With this in mind, methods used for sex-determination and disposal would be best employed before the embryo feels pain, approximately 7 days before nociception appears [START_REF] Eide | Development of the longitudinal projection patterns of lumbar primary sensory afferents in the chicken-embryo[END_REF]. A consensual limit on 9 days of incubation has been proposed as there is a controversial grey zone for up to 15 days depending on studies.

In ovo sexing methods are based on the initial postulate that male embryos and female embryos exhibit specific features (anatomical, physiological, molecular and genetic) that should allow for the discrimination between sexes during incubation of fertilized eggs. Some of these methods are universal while others have been specifically designed for selected genotypes. A review of the various in ovo sexing techniques that were close to market was published in 2018 [START_REF] Hein | Egg sexing close to market[END_REF]. Of these, only two are commercialized and in use by the poultry sector (Seleggt 2019, Plantegg 2020). Some require the sampling of embryonic cells or embryo-derived cells while others rely on the sampling of extra-embryonic fluids such as the allantoic fluid.

Dimorphic chromosomes

In birds, unlike mammals, males are homogametic (two Z chromosomes), whereas females are heterogametic (Z and W chromosomes). The constraint of techniques built upon these sexual characteristics is the sampling of embryo-derived cells that bear the embryonic genome. The detection of W-or Z-specific genes by polymerase chain reaction to distinguish female embryos from male embryos is well established [START_REF] Clinton | A rapid protocol for sexing chick embryos (Gallus g. domesticus)[END_REF][START_REF] Ellegren | First gene on the avian W chromosome (CHD) provides a tag for universal sexing of non-ratite birds[END_REF][START_REF] Smith | The avian Z-linked gene DMRT1 is required for male sex determination in the chicken[END_REF]). The German company PLANTEGG has developed a PCR-based method using a few drops of the allantoic fluid that contain embryonic cells [START_REF] Plantegg | In-Ovo Gender determination[END_REF].

Another approach is based on the analysis of the DNA content of the embryonic cells, considering that the Z chromosome has 200% more DNA content than the W chromosome [START_REF] Mendonça | DNA content differences between male and female chicken (Gallus gallus domesticus) nuclei and Z and W chromosomes resolved by image cytometry[END_REF]) and male cells containing the ZZ chromosomes are about 2% bigger than female ZW containing cells. Using infrared spectroscopic imaging on fertilized eggs prior to incubation, [START_REF] Steiner | Gender determination of fertilized unincubated chicken eggs by infrared spectroscopic imaging[END_REF] corroborated that male blastoderm cells have a higher DNA content than female blastoderm [START_REF] Steiner | Gender determination of fertilized unincubated chicken eggs by infrared spectroscopic imaging[END_REF]. Using this method, gender determination is possible at very early stages, is rapid (few seconds) and accurate. However, it requires germinal disc sampling with possible long-term effect on the development of the embryo or the chicks after hatching. Some other authors also use the length of the sex chromosomes to develop in ovo sexing methods based on Raman spectroscopy [START_REF] Galli | In Ovo Sexing of Domestic Chicken Eggs by Raman Spectroscopy[END_REF][START_REF] Galli | In ovo sexing of chicken eggs by fluorescence spectroscopy[END_REF], Galli et al. 2018). These authors focused on bloods cells, all of which are nucleated in birds, and analyzed the spectra of blood fluorescence.

Differentiation between males and females was shown to be 90% from the 4th day of incubation. According to the authors, this technique has no visible effect on the hatched chick, but decreased the hatchability by about 10%. The invasiveness was further reduced by keeping the eggshell membrane intact [START_REF] Galli | Sexing of chicken eggs by fluorescence and Raman spectroscopy through the shell membrane[END_REF]) leading to the development of a prototype (Muller-Niegsch 2017). However, there is no current information on the state of progress in terms of commercialization. Similarly, the AAT group developed a Raman-spectroscopic method that would allow for sexing eggs after 4 days of incubation. This approach remains semi-invasive as a small piece of the eggshell needs to be removed to access the embryo and the surrounding yolk sac vascularization, prior to Raman spectroscopy measurements (AAT 2020).

Dimorphic compounds

Besides chromosome-based strategies, techniques that use reported differences in the hormonal and metabolic status between male and female embryos have been proposed. In 2013, Weissman et al., published that the allantoic fluid from female embryos displayed significantly higher estrone sulphate (female hormone) levels than males and that this difference was detectable as soon as 9 days of incubation [START_REF] Weissmann | Sexing domestic chicken before hatch: a new method for in ovo gender identification[END_REF]. The discovery led to the development of a prototype by SELEGGT with a sexing accuracy of 97% (Seleggt 2019). The eggs resulting from this approach are called "respeggt" eggs and were successfully introduced in 2018 in Germany. They are available in boxes of six at all 5 500 REWE and PENNY stores.

For all these techniques, sampling embryonic-derived cells or extraembryonic fluids implies invasive or semi-invasive technique (egg opening/eggshell drilling) that would increase the risk of low viability/impaired hatchability afterwards. A non-invasive technique would likely supplant all of the above approaches.

Besides hormones, it has been shown that glucose, choline and some amino-acids (valine) are more concentrated in allantoic fluid from females [START_REF] Bruins | Gender, viability and/or developmental stage determination of avian embryos in ovo (NL)[END_REF] while the same authors also found that butylated hydroxytoluene is a particularly relevant volatile dimorphic biomarker [START_REF] Bruins | Method and system for the nondestructive in ovo determination of fowl gender (NL)[END_REF].

Such dimorphic volatile compounds are promising as they can diffuse through the eggshell pores and may be detectable at the surface of the eggshell [START_REF] Webster | Avian egg odour encodes information on embryo sex, fertility and development[END_REF][START_REF] Costanzo | The odour of sex: sex-related differences in volatile compound composition among barn swallow eggs carrying embryos of either sex[END_REF][START_REF] Knepper | System and method for in ovo sexing of avian embryos[END_REF]. Taken together, these data were probably the initial step for the development of a prototype by In Ovo, a spin-off of Leiden University (InOvo 2020). Since 2017, they have been developing their current Alpha prototype, realizing a throughput of 1 800 eggs per hour with 95% accuracy. In the InOvotive project that started in June 2020 (CORDIS 2020), they plan to scale this prototype to reach more than 10 000 eggs per hour to meet hatchery needs.

Spectral dimorphism of the whole egg

The most promising non-invasive technologies developed to date are based on spectroscopic methods applied to the whole egg, such as hyperspectral imaging (Canadian Hypereye company and Agri Advanced Technologies (AAT, Germany)) and the combination of spectroscopy and biosensors (SOO project, French company Tronico and the French National Centre for Scientific research). The technology of Hypereye uses hyper-spectral technology to acquire a specific signature through mathematical algorithms to determine the gender of the embryo from the day of lay onwards. Commercialization date and progress are not known. Several announcements had been made and a prototype was expected for 2018, with a throughput of 50 000 eggs per hour. However, since then, there is no public information about their progress in this field. A similar strategy has been developed by AAT technology (AAT 2020). Using a specific genotype (Lohmann Tierzucht GmbH) where female chicks have brown down feathers and the males have yellow down feathers, Gohler et al ( 2017) describe a non-destructive optical method for sex determination. This hyperspectral method has been shown to reach 97% efficiency between 11 and 14 days of embryonic development and is expected to be implemented to allow the sexing of eggs at earlier stages (7 th day of incubation).

According to the website (AAT 2020), the technique is 95% accurate and 20 000 eggs can be tested per hour per machine. Hyperspectral measurement technology has been improved for large-scale practical use and French egg suppliers have started to use this technology since the beginning of 2020.

In 2017, French Agriculture Minister Stéphane Le Foll granted Project SOO ("Sexage des Oeufs d'Oiseaux" in French or "sexing avian eggs") with 4.3 million euros to finance the development of new in ovo sexing methods. This project, with the French Tronico company and the French National Centre for Scientific research, focuses on a method that combines spectroscopy (response to a light pulse) and the use of biosensors, that it is claimed will be 90% reliable in sexing eggs as soon as 9 days of incubation. The prototype was initially expected at the end of 2019. Although the current technologies and prototypes need to be improved, it has to be mentioned that ovosexing provides another advantage as compared with current manual methods for sexing as it will avoid the manipulation of chicks at hatch and thus will limit additional stress for animals. They may also be transposed to other bird species of industrial interest, such as foie-gras production where only males are reared (female ducks are culled at hatch as their liver is too small and contains many veins).

Sex manipulation by genome editing

Although the social acceptability of such approaches remains very poor [START_REF] Gremmen | A Public Survey on Handling Male Chicks in the Dutch Egg Sector[END_REF], the advent of CRISPR/Cas9 genome editing highlights a new opportunity to potentially generate a gender that would bear a specific marker, which would increase the feasibility to detect males from females at soon as the egg is laid, or to create all-female or all-male progeny using some of the imaging approaches outlined above. In this regard, the program EggXYT developed a gene edited breed of chickens based on the introduction of a fluorescent marker within the sexual chromosome (eggXYt). Males can be detected using fluorescence imaging and reliability is claimed to be 100%. Its state of commercial development is at the level of prototype 3.0, which does not yet appear fast enough for high throughput hatcheries.

There are also many challenges regarding the genetic technologies that would allow the production of single-sex litters [START_REF] Douglas | Advances and challenges in genetic technologies to produce single-sex litters[END_REF]. Although gene-drive methods can also have disadvantages (mutation, abnormalities, uncontrolled spread of synthetic gene drives), some consumers may consider the use of transgenic animals in agriculture ethically preferable to the culling of the unrequired sex [START_REF] Douglas | Advances and challenges in genetic technologies to produce single-sex litters[END_REF]. This approach, although still unacceptable by most public authorities, still elicit interests and scientific research/development. Thus, there is an urgent need to discuss more globally about the acceptability and the potential benefit/risk balance of these genetic methodologies.

Dual purpose breeds and/or growing layer male chicks

The development or revival of dual-purpose strains, with females producing eggs and males producing enough quality meat to be marketed, is currently being examined and will likely form a segment of the future market. If killing male day-old chicks is not seen as an ethically defensible position, then the use of dual-purpose chickens seems a straightforward proposition. Even if male chicks are killed humanely and they are consumed, albeit for pet and zoo animals, the industry struggles with the ethics of producing animals that do not live a full life [START_REF] Bruijnis | Moral "lockin" in responsible innovation: The ethical and social aspects of killing day-old chicks and its alternatives[END_REF]. The dualpurpose chicken allows the male to be reared for meat production, growing faster with more saleable meat than the male of chickens bred purely for laying. Currently in the available laying strains the growth of males does not meet the requirements for the production of quality meat at a competitive cost [START_REF] Koenig | Utilization of laying-type cockerels as coquelets: Influence of genotype and diet characteristics on growth performance and carcass composition[END_REF][START_REF] Gremmen | A Public Survey on Handling Male Chicks in the Dutch Egg Sector[END_REF]). However, even if the resulting meat is comparable with meat from broilers, the production remains less competitive in economic terms, but also in terms of resources and environmental pollution [START_REF] Koenig | Utilization of laying-type cockerels as coquelets: Influence of genotype and diet characteristics on growth performance and carcass composition[END_REF]. A number of approaches have been adopted to produce dual-purpose birds, from the use of lines of chicken that have served traditional markets to specific breeding programmes, which produce chickens where the males can almost compete with slow growing breeds of broiler chicken, although still often with 10% less meat product. In many cases, these are a cross between broiler and layer parent stock. In general, although the males take longer to get to a reasonable slaughter weight than a slow growing broiler, meat quality and acceptance seem to compare well with broiler meat [START_REF] Mueller | Carcass and meat quality of dual-purpose chickens (Lohmann Dual, Belgian Malines, Schweizerhuhn) in comparison to broiler and layer chicken types[END_REF].

The amount of leg meat is larger in dual purpose breeds but this can be favourable in some markets. The main disadvantage of dual-purpose breeds is the lower yield in breast meat, therefore dual-purpose breeds are often sold as a complete carcass.

The same acceptance is true for the eggs from the females, which compare favourably in some studies with the possible exception that brown egg dual-purpose breeds produce eggs that are lighter than from a pure brown egg layer. However, there are reports of poorer egg quality in some lines, in both external (shell strength, shell color) and internal (Haugh units, blood and meat spots) parameters. There may be other benefits of the dual-purpose bird as there is some evidence that dualpurpose chickens suffer less from issues such as mortality from injurious pecking.

The real issue comes from the economics and environmental impact of the dualpurpose breeds; indeed, it has been suggested that the current system that has evolved has produced a 'lock in' [START_REF] Bruijnis | Moral "lockin" in responsible innovation: The ethical and social aspects of killing day-old chicks and its alternatives[END_REF]. Essentially the current production system has developed to be so efficient with a comparatively small environmental impact that it is impossible for any competing systems to be established, either ethically or indeed economically. The biological and economic consequences for different options of dual-purpose chickens have been examined in several studies [START_REF] Leenstra | Killing day-old chicks? Public opinion regarding potential alternatives[END_REF]. This demonstrated that dual purpose chickens might serve as a niche market, but a total shift to dual purpose chickens in order to solve the problem of killing day-old males would not be realistic when looking at the environmental burden and the economics. Alternative systems such as dual-purpose chickens have higher environmental costs, taking more resources to produce the same amount of food, with ratios for the conversion of feed to meat lying above four, while for broilers it can be around 1.6 [START_REF] Giersberg | Rearing Male Layer Chickens: A German Perspective[END_REF]. Egg production from dual-purpose hens also has greater environmental impact as they lay typically around 50 fewer eggs in a year but consume similar or more food to do so. It is difficult to argue that the system is ethically superior compared to current strains if negative environmental impacts increase. It is argued that the 'Responsible Innovation' approach, which balances economic, socio-cultural and environmental aspects of any new system, would need to be promoted to shift production and get consumers to accept on a larger scale the products from dual purpose rearing systems or, indeed, any alternative system that improves the ethical dimensions of production [START_REF] Bruijnis | Moral "lockin" in responsible innovation: The ethical and social aspects of killing day-old chicks and its alternatives[END_REF]. In the absence of legislative changes, the use of dual-purpose hens will likely remain an expanding but niche product requiring strong marketing [START_REF] Busse | Ethical Concerns in Poultry Production: A German Consumer Survey About Dual Purpose Chickens[END_REF].

A German dual-purpose initiative, which includes the rearing of day-old layer male chicks is again working in a niche market, where an increasing number of day-old males are kept for meat production. In Germany, they have introduced this as the Bruderhahn (Brother cockerel) initiative (BID 2020). Eggs are sold for a premium price in order to compensate for potential economic losses when growing the males.

Because of their inefficient feed conversion, and different characteristics (they differ in breast meat yield, taste and tenderness compared to conventional broilers), the cost of production is higher with a smaller market. It is forecast that the entire organic egg market will adopt the principle of growing the male layer chicks, as they do not see in-ovo sexing as an acceptable solution, as the male embryos are still discarded.

A specific example of a system utilising 'Les Bleues' chicken has been used in the 'ei care' project in north eastern Germany (eiCare). Both males and females are raised for meat and eggs, respectively and are marketed with the 'ei care' branding to organic shops and supermarkets. The project is a partner in a research program for sustainable development and currently has 4 farms producing eggs and meat sold relatively locally.

There is an urgent need for research and development of new dual-purpose strains by crossing selected genotypes to optimize their productivity under realistic farm conditions as well as optimising the quality of derived products (eggs and meat). It is also necessary to understand the behaviour of these new strains in different farming systems and under different environmental conditions, all of which should lead ultimately to the best scenario/trade-off in terms of health for the hen and of costs for farmers. To complement this, there is also a need to understand consumer attitudes to the proposed systems. It is also important to consider that some consumers prefer brown eggs while others prefer white eggs (cultural habits). It is forecast that we will need to have several crosses as options, depending on countries, to meet peoples cultural requirements.

Towards more ethical animal husbandry?

Already there are some well developed systems that are marketing the concept.

Poulehouse is a company founded by Fabien Saullman, Elodie Pellegrain and Sébastien Neuch (Poulehouse). The slogan of this company is "the egg that doesn't kill the hen". They define themselves as "A responsible production method. Ethical.

Innovative. From production to the plate". Classically, hens are slaughtered at 70-80 weeks when their productivity becomes uneconomic. Poulehouse offers a rearing method where the hen is kept alive until its natural death, which can occur at 7-12 years of age. The hens produce shell eggs of sufficient good quality for about 3 years, which are sold at a price of about 1 euro per egg, which is 3 times more expensive than organic eggs. The selling price of the eggs thus makes it possible to house and feed the non-producing hens until their natural death. This method of production generates a number of zootechnical constraints such as the control of moulting. Indeed, a hen after 15-16 months of production undergoes a moult that is characterised by a regeneration of the reproductive tissues. One month after the start of moulting, the hen will again lay good quality eggs, but the laying time will rapidly decrease as the cycles progress. This moult may be caused by a decrease in light and energy rationing strategies in the feed. Poulehouse objective is to achieve 3 moulting periods separated by production periods of 9-12 months. At the moment, the oldest flock is 3-4 years old. Health management of older flocks will also be a challenge when the flocks will be at the end of their life.

Poulehouse produces organic eggs (code 0), but also free-range eggs (code 1).

Poulehouse has initiated a collaboration with the German start-up company Seleggt, which has developed a technique to detect the sex of the chick in the egg and thus hatch only females (see the paragraph "sexing eggs"). Two of their farms are already producing eggs that do not kill either the hen or the male chick. Eventually, they want to generalise this process to produce "eggs that do not kill the hen and the male chick". The Poulehouse company has undertaken numerous marketing campaigns to promote their products. It is still a weak market, but it does reflect a trend of producers to serve consumers who want to consume products in accordance with their convictions.

Another initiative can be found in the Netherlands, which is the Kipster farm concept (Kipster). In this concept it is all about sustainability, and they try to include all elements with respect to sustainability, not only the ethics involved, but also with a large focus on the impact of farming on the environment. At the Kipster farm, they focus on closed loop farming, and they try to limit the waste generated during the production process. Together with LIDL supermarkets, they have developed new products based on the meat coming from the processed spent hens and layer males. This allows them to create value out of the products that would otherwise be considered as waste/products with low economic value. The eggs are sold with a premium price, and no involvement of an egg packing station; in this way the egg producer can benefit more from exploiting this innovative concept. High standards of animal welfare are combined with extremely transparent farming. This to reduce the growing gap between producers and consumers, and educate the consumers on the origin of their food. At Kipster they have deliberately chosen white egg layers, as they have a lower ecological footprint compared to the brown egg layer [START_REF] Mollenhorst | The contribution of breeding to reducing environmental impact of animal production[END_REF]. The main reason for this lower ecological footprint in white egg layers is their ability to be kept for longer production periods compared to the brown egg layer, resulting in more saleable eggs produced per hen housed, higher total egg mass produced and better feed efficiency. That the popularity of the white egg layers is on the rise can be clearly seen in the Netherlands, where the brown to white egg layer ratio went down from 60 -40 in 201260 -40 in , to 35 -65 in 201860 -40 in (IEC 2018).

In the UK, because the free-range concept has been around for so long, there are considerable challenges to any other systems entering the market. Free-range is the mainstream product which customers identify as an ethical choice and to some extent this may be preventing new systems being produced, perhaps another example of a 'lock in'. Consumers identify with what free-range means. Although there might be advantages in terms of health for the hens with something as simple as a barn system for example, it has had very limited success as a product. More ambitious examples of sustainable systems as outlined in France, Netherlands and Germany have not yet emerged commercially to our knowledge.

Conclusion

In the next decade, the egg sector will have to deal with the evolution of the systems of egg production in cages vs non-cages to consider welfare and sustainability. We have described recent developments in science, technology and production strategies that are intended to tackle ethical issues in layer hen systems. They included the extension of the laying period and the development of more recent production methods that try to be ethical. Another important challenge for the sector is the use of alternatives to the culling of male day-old chicks of layer lines. The development of genotypes to obtain dual-purpose strains is in progress, but the current genotypes do not yet meet the requirement for the production of quality meat and egg. Introducing in ovo sexing techniques in hatcheries implies additional costs for producers who will have to reorganize logistics, but also for consumers who will pay more for eggs. Because of the drive to achieve the goal of all-female chicks, in addition to properly researched proposals rooted in biology, the field has attracted some unlikely 'snake oil' solutions which industry and funders should be aware of. To date, most technologies are not efficient to determine the sex of the embryo at the day of lay. They require 1) the removal of eggs from incubators for sexing, 2) reincubation of female eggs potentially increasing the risk of embryonic mortality 3) the elimination and new valorization of male eggs as high-quality feed (SELEGGT option) or other uses, and the management of male chicks if the method is not 100% reliable.

In the future, the accuracy of methods may be greatly improved by combining several dimorphic features to get to the level of accuracy desired of near 100% and by using artificial intelligence tools to integrate data. However, it is likely that if the method selected is not 100% accurate, chicks will have to be re-examined by sexers at hatch to avoid the introduction of males in female flocks. Already in ovo immunisation is performed routinely, which suggests it is possible to do at scale without detriment. In parallel, several strategies based on sex manipulation (transgenesis, genomeediting) have shown a high potential and efficiency but the ethical and social acceptability of such approaches remains very poor. Although consumers are willing to pay more for eggs from non-cage systems in many parts of Europe, the more widespread adoption of the systems described in this review will increase the cost of eggs and egg products.

It should be noted that the solutions are still very marginal in terms of production or remain untested and are not the dominant production model. No one knows whether these modes of production will expand or remain a niche market in the future. What also seems imminent, is there will be increased costs associated with the change that may only partly be offset by increased efficiency. Most alternatives for sexing are predicted to result in an extra-cost of 1 to 5 cents per egg for consumers. This makes investment decisions difficult for farmers. However, it seems that some of the changes are inevitable, at least in European markets.

Figure legends

Figure 1 :

 1 Figure 1: Percentage of laying hen numbers by production systems in 2019, in

Figure 2 :

 2 Figure 2: Egg consumption in France in 2018 by distribution channel (ITAVI, 2019).

Figure 3 :

 3 Figure 3: Schematic representation of the effect of increasing the laying cycle on

Joël Gautron: 0000-0001-7800-0578 Sophie Réhault-Godbert: 0000-0003-2800-3417 Ian Dunn: 0000-0003-3630-0120 Teun Van De Braak : None Acknowledgements A part of this bibliographic review was realized within the framework of a scientific and collective expertise (ESCo INRAe) on the quality of food of animal origin according to production and processing conditions. The bibliographic review related to "Alternatives to the culling of one-day-old male chicks" was realized under the framework of the Poultry and Pig Low-input and Organic production systems' Welfare (PPILOW) program. Part of this review was written in close cooperation with Institut de Sélection Animale B.V., a Hendrix Genetics Company. Financial support statement JG and SRG were supported by the European Commission (PPILOW project, grant Number 816172). ICD and the Roslin Institute are funded by a BBSRC Institute strategic program (grant BB/P013759/1). ICD is also funded by the Foundation for Food and Agricultural Research (grant ID 550396) ICD and TVB are part of a COST action CA15224 Keel Bone Damage.

Data and model availability statement

No data or model were generated as part of this study.

Ethics approval

This review did not require any animal handling or procedures.

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